2007 Wyman Ann Rev Gen ds DNA rep

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DNA Double-StrandBreak Repair: Alls Wellthat Ends Well

Claire Wyman and Roland Kanaar

Department of Cell Biology & Genetics and Department of Radiation Oncology,Erasmus MC, 3000 DR Rotterdam, The Netherlands; email: r.kanaar@erasmusmc

Annu. Rev. Genet. 2006. 40:36383

First published online as a Review inAdvance on August 8, 2006

The Annual Review of Geneticsis online at

http://genet.annualreviews.org

This articles doi:10.1146/annurev.genet.40.110405.090451

Copyright c 2006 by Annual Reviews.All rights reserved

0066-4197/06/1215-0363$20.00

Key Words

DNA damage, genome (in)stability, homologous recombination

nonhomologous DNA end-joining, stalled DNA replication

AbstractBreaks in both DNA strands are a particularly dangerous thrto genome stability. At a DNA double-strand break (DSB), pote

tially lost sequence information cannot be recovered from the sa

DNA molecule. However, simple repair by joining two broken en

though inherently error prone, is preferable to leaving ends b

ken and capable of causing genome rearrangements. To avoid DS

induced genetic disinformation and disruption of vital process

such as replication and transcription, cells possess robust mec

nisms to repair DSBs. Because all breaks are not created equal, t

particular repair mechanism used depends largely on what is p

sible and needed based on the structure of the broken DNA. Wargue that although categorizing different DSB repair mechanis

along pathways and subpathways can be conceptually useful, in ce

flexible and reversible interactions among DSB repair factors form

web from which a nonpredetermined path to repair for any num

of different DNA breaks will emerge.

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DSB: DNAdouble-strand break

Contents

DIFFERENT MEANS TO THE

ENDS . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

ALL ENDS ARE NOT CREATED

EQUAL . . . . . . . . . . . . . . . . . . . . . . . . . 364

ONE END OR TWO . . . . . . . . . . . . . . 367

REJOINING OF TWO-ENDEDDSBs BY THE CORENONHOMOLOGOUS DNA

END-JOINING

CO M PO N E N T S . . . . . . . . . . . . . . . . 3 6 7

END CLEANING BEFORE

JOINING . . . . . . . . . . . . . . . . . . . . . . . 369

WHEN RECOMBINATION IS

NEEDED AN ORDERED

SERIES OF REACTIONS ISSET IN MOTION . . . . . . . . . . . . . . 369

FIRST, KEEP REPAIR PARTNERSCLOSE THROUGH ALL THE

PRELIMINARY STEPS . . . . . . . . . 370

SECOND, CREATE

SINGLE-STRANDED DNA AT

THE BREAK . . . . . . . . . . . . . . . . . . . . 371

THIRD, ASSEMBLING

NUCLEOPROTEINFILAMENTS, THE CORE

RECOMBINATION

CATALYST . . . . . . . . . . . . . . . . . . . . . . 372

AFTER THE RECOMBINATIONPARTNERS ENGAGE . . . . . . . . . . 374

BEFORE THE BEGINNING AND

AFTER THE END . . . . . . . . . . . . . . 374

BEFORE RECOMBINATION. . . . . . 375

AFTER STRAND EXCHANGE . . . . 375SUMMING UP AND MOVING

O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 7 6

DIFFERENT MEANS TO THEENDS

DNA double-strand breaks (DSB) are in-

teresting DNA lesions because they can be

both detrimental and beneficial to organisms.

DSBs, such as those induced by exogenous

DNA-damaging agents or endogenously pro-

duced reactive oxygen species, can promo

genome rearrangements that initiate carcin

genesis or apoptosis (29). By contrast, DS

can actually be beneficial when they occ

in a controlled manner in the context specialized events that demand that genom

sequences be rearranged, such as during d

velopment of the immune system and gen

ation of genetic diversity in meiosis (52, 7

108). To counteract the deleterious effects

unwanted DSBs and to promote the ben

ficial effects of programmed DSBs, multi

DNA repair mechanisms have evolved (9Two distinct DSB repair mechanisms, nonh

mologous DNA end-joining and homologo

recombination, are schematically depicted

Figures 1 and 2, respectively. Nonhomo

gous DNA end-joining uses extremelylimi

or no sequence homology to rejoin juxtapo

ends in a manner that does not need to

error free (67). Homologous recombinati

requires extensive regions of DNA homoloandrepairs DSBs accurately by using inform

tion on theundamaged sisterchromatid (11

These distinct DSB repair mechanisms can

subdivided further, depending on the natu

of the DNA end. Additional discussions

the details of DSB repair mechanisms can

found in a number of reviews (15, 39, 47, 6

ALL ENDS ARE NOT CREATEDEQUAL

The current mechanistic picture of DSB

pair has been cobbled together from expe

mental systems that span the methodologi

spectrum from biochemical analysis of li

ited but defined components to genetic an

ysis in animals and cells. Knowing what stru

tures are created when DNAis experimentabroken is an essential prerequisite for sorti

through the results. For biochemical analy

the DNA substrates to be repaired are crea

to mimic a specific in vivo situation and

usually defined sequences and structures. T

study repair in cells or animals, DSBs are

duced by a variety of treatments that differ

the type and amount of breaks induced as w

364 Wyman Kanaar


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Break formation

End binding; Ku70/80

End juxtaposition; Ku70/80, DNA-PKcs

No end processing required

XRCC4

DNA ligase IV

XLF

Artemis

TdT

pol lambda

pol mu

End processing required

XRCC4

DNA ligase IV

XLF

Figure 1

DNA double-strand break repair through nonhomologous DNA end-joining. The formation of atwo-ended DSB, for example, by ionizing radiation, is indicated. The DNA ends are substrates forbinding of the Ku70/80 heterodimer, which localizes DNA-PKcs to the ends and promotes theirjuxtaposition. If no further processing of the ends is required, the additional core components ofnonhomologous DNA end-joining, XRCC4, DNA ligase IV, and XLF can complete the rejoiningreaction. Alternatively, end processing may require the activities of the nuclease Artemis and/or the DNApolymerases TdT, pol lambda, and pol mu. The Ku heterodimer likely plays a central role inorchestrating the activities of the proteins involved in nonhomologous DNA end-joining. Transientreversible interaction of the processing factors with the core components provides great flexibility in thecombination of broken ends that can be rejoined because this does not require a strict order in which theprocessing factors engage or in which the four strands will be processed.

as in the amount of collateral damage in the

form of other types of DNA lesions. For in-stance, in cell biology experiments, DSBs can

be introduced directly by ionizing radiation,

using an X-ray machine or a 137Cs course, by

synchrotron-generated ultrasoft X rays (63),

by -particles (3), or by heavy ion irradia-

tion (31, 45). All of these methods certainlyinduce DSBs but also other types of lesions

such as single-strand breaks, damage to the

sugar moietyand base modifications(22). Fur-

thermore, radiation-introduced DSBs are not

clean in that the DNA ends created oftencannot be directly ligated. Thus mechanisms

for repairing these types of breaks will nec-

essarily include DNA end-processing steps as

a prerequisite to their repair, irrespective ofthe DSB repair mechanism used. Popular but

less well-defined methods to introduce DSBs

NonhomologousDNA end-joininrejoining of ends

from a broken DNmolecule withoutuse of a repairtemplate. The DNends may beprocessed to expoor create ligatableends, a 3-hydroxyand a 5-phosphatSequenceinformation can blost upon rejoininmaking this proce

error proneHomologousrecombination:exchange ofbase-paired partnbetween twohomologous DNAmolecules

involve laser illumination with or without ad-

dition of an activating compound associatedwith the DNA such as UV-A laser light fol-

lowing incorporation in DNA of halogenated

thymidine analogs (45, 46, 93) or incubation

of the cells with Hoechst dye (7, 76, 103),

irradiation with a pulsed neodymium-doped

yttrium aluminum garnet laser (37, 38) andpulsed multiphoton laser (55). Again, breaks

are certainly produced, but an unknown spec-

trum of other lesions also results. Thus, DNA

repair occurring in cells subjected to these

treatments must deal with a variety of lesions,sometimes undefined, and often with an arti-

ficially high local damage load. In addition,

a different chemical structure at the breaknecessitates specific processing steps for re-

pair that will depend on the way damage was

created.

www.annualreviews.org DNA Double-Strand Break Repair 365


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a

b

c

d

e

g

f

h

i

j

Figure 2

DNA double-strand break repair through homologous recombination. The close pairs of parallel linesrepresent the two strands of duplex DNA, while the lighter colored pair is the sister chromatid of thedarker colored pair. The left-hand side of the top most strand has 3 polarity. (a) One of the two sisterchromatids has suffered a DSB. (b) Processing results in single-stranded tails at the break with3-hydroxyl ends. The tails are a substrate for nucleoprotein filament formation with a recombinase.(c) Nucleoprotein filament directed homology recognition and DNA strand exchange lead to jointmolecule formation between the broken DNA and the intact sister chromatid, resulting in a structureknown as a D-loop. (d) In one model, termed DSBR, DNA synthesis (indicated by the arrowhead) fro

the D-loop intermediate and migration of the junction lead to formation of a Holliday junction andengagement of the second end of the break with the intact sister chromatid. (e and f) Continued DNAsynthesis followed by strand ligation results in a double Holliday junction intermediate. (g) Resolutionthis intermediate by junction resolvases can result in crossover and noncrossover recombinants.Alternatively, the intermediate can be resolved by the combined action of a helicase and a topoisomerI, which gives rise to noncrossover recombinants exclusively. (h, i, and j) As an alternative to the DSBRmodel, the synthesis-dependent strand annealing (SDSA) model proposes that after DNA synthesis frthe D-loop intermediate, the newly synthesized strand is exchanged between the sister chromatidtemplate and the other end of the processed break. This will result in noncrossover recombinants.

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On the other hand, breaks with defined

structure, genomic location, and number can

be created by enzymes, either rare cutting

restriction enzymes such as I-SceI, the HO

endonuclease of Saccharomyces cerevisiae, orchimeric enzymes that couple the nuclease

domain of the type II restriction enzyme Fok

I to Zn-finger DNA-binding domains (27, 32,

71). DNA cut by enzymes often has comple-

mentary single-strand overhangs that can be

easily ligated and do not require otherwise

essential steps for processing authentic dam-

age. Another special class of defined DSBs

results in covalent protein-DNA complexes,such as those induced by topoisomerase in-

hibitors (4, 70). The resulting protein-bound

DNA ends may be specifically recognized and

must be specifically processed before rejoin-

ing or recombination can proceed. Thus dif-

ferent enzymaticactivitiesare expected to par-

ticipate based on the type of damage being

studied, though all are often referred to sim-ply as DSBs.

ONE END OR TWO

Break repair possibilities are influenced by

the manner in which the break is created.

DSBs are caused directly, for instance, by ion-

izing radiation, by severing both phosphate-

sugar backbones close together. These twoends can, in principle, be rejoined by non-

homologous DNA end-joining (Figure 1) or

be repaired by homologous recombination

(Figure 2) with their intact sister chromatid

as therepair template.More commonly, DNA

breaks occur indirectly as a result of damage

or discontinuities in one strand encountered

during replication (15). Here often only one

free end is produced (Figure 3g), which elim-inates nonhomologous DNA end-joining as a

means for repair and therefore requires ho-

mologous recombination (16). During repli-

cation a homologous duplex, in the form of

the sister chromatid, is close by and available

as a recombination partner because daugh-

ter duplexes are linked at the replication fork

(Figure 3). On the other hand, when a DNA

Replication forksite along a DNAmolecule wherereplicative DNAsynthesis is takingplace. The DNA

structure formed be drawn as a forkjunction, where thtwo parental stranare still a pairedduplex beforesynthesis, separatiinto the newlysynthesized daughduplexes. Theassociated proteinminimally includehelicase, a primas

and both leading alagging strand DNpolymerases.Leading and laggistrand synthesis arcoupled andtherefore occur incoordinated fashioat a fork

break is created directly, both ends are in close

proximity, which favors their repair by non-

homologous DNA end-joining over homol-

ogous recombination because a homologous

sister chromatid may not be near. Thus itfollows logically that homologous recombi-

nation is mostly limited to S phase, whereas

nonhomologous DNA end-joining can oper-

ate irrespective of the cell cycle phase.

REJOINING OF TWO-ENDEDDSBs BY THE CORE

NONHOMOLOGOUS DNAEND-JOINING COMPONENTS

The simplest way to heal a two-ended DSB

is to ligate it back together through nonho-

mologous DNAend-joining (107). Thestruc-

ture of the DSB end will direct the sub-

strate into differential use of end-processing

factors. A clean two-ended DSB, with ei-

ther blunt ends or small 5 or 3 complemen-

tary overhangs (6), is a substrate for a non-homologous DNA end-joining reaction that

requires just its core components: Ku70,

Ku80, DNA-dependent protein kinase cat-

alytic subunit (DNA-PKcs), XRCC4, XLF,

and DNA ligase IV (Figure 1). The Ku70

and Ku80 proteins form a heterodimer that

displays affinity for DNA ends. X-ray crys-

tallography revealed that the Ku70/80 het-erodimer forms a ring with a hole that fits

DNA, explaining its preference for binding

DNA ends (102). Ku70/80 is thought to re-

cruit DNA-PKcs. The precise role of DNA-

PKcs is not clear, but its association near ends

might be importantfor their juxtaposition (19,

85). In addition, DNA-PKcs binding causes

Ku70/80 to move about one helical turn in-

ward from the end (118), thereby facilitatingaccess of other proteins to the business end of

thebreak. In particular, thefinal step in rejoin-

ing is mediated by DNA ligase IV (41). This

ligase is associated with a dimer of XRCC4

(57). XRCC4 can bind to DNA, is required

for the stability of DNA ligase IV in vivo,

and stimulates its adenylation and ligase activ-

ity (8, 25, 26, 56). Recently, an XRCC4-like



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e

a

b

c d

f

Figure 3

Re-establishing DNA replication forks upon encountering DNA damage in the leading strand templat(a) Shown is a replication fork with leading and lagging strand DNA synthesis. Arrowheads indicate3-hydroxyl ends. The small circle represents DNA damage in the leading strand template.(b) Uncoupling of DNA polymerases when a forks encounters the damage results in continued laggingstrand synthesis, creating a single-strand region on the leading strand template. ( c) DNA synthesis can

restarted by de novo synthesis of a primer downstream of the damage in the template, leaving the lesioin a single-stranded gap that might require recombination with the sister chromatid, translesion DNAsynthesis or their combination for its conversion to double-stranded DNA. (d) Alternatively, the lesiocan be bypassed through homologous recombination directly. The blocked nascent strand can invade intact sister chromatid to form a D-loop. (e) D-loop extension by DNA synthesis can lead to formationa Holliday junction when the displaced lagging strand template pairs with the leading strand template(f) Branch migration of the Holliday junction in the direction of fork movement creates an intermedifrom which an active replication fork can be reassembled. (g) In case the lesion in the template DNA isingle-strand nick, the replication fork will be converted into a one-ended DSB. (h) Processing of the eresults in single-stranded DNA that can invade the sister chromatid via a D-loop intermediate fromwhich DNA synthesis will result in the arrangement of DNA strands depicted. (i) Holliday junctionresolution will result in an intermediate onto which a competent replication fork can be assembled.(j) The structure shown is redrawn from panel (i) to more clearly indicate the fork reassembly potentof the intermediate.

protein, XLF (also known as Cernunnos), has

been identified as an interaction partner of

the DNA ligase IV/XRCC4 complex (1, 9).

Its function in nonhomologous DNA end-

joining has not yet been defined, but cells

from patients with mutations in the XLF

gene are radiosensitive and DSB repair d

fective. The patients themselves are immun

deficient because of their inability to pro

erly process the DSB intermediates requir

for the assembly of active immunoglobu

genes.

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END CLEANING BEFOREJOINING

DSBs differ not only with respect to the num-ber of DNA ends but also in the chemical

composition of the ends. For example, ion-

izing radiation-induced DNA breaks are not

directly ligatable because they are not proper

substrates for DNA ligases, which require 3

-hydroxyl and 5-phosphate groups. In addi-

tion to damaging the phophodiester back-

bone, reactive oxygen species resulting from

radiation cause base and sugar damage. Thus,many radiation-induced DNA ends require

nucleolytic processing and DNA synthesis to

remove and replace nonligatable remnants

of nucleotides and incompatible single-strand

overhangs. Obviously, the core nonhomol-

ogous DNA end-joining reaction described

above will not suffice for repair of these DNAends. Genetic and biochemical experiments

have revealed the existence of activities thataugmentthecorereactionsothatitcanhandle

DNA ends with a variety of different chemical

and secondary structures (Figure 1).

The Artemis protein is a versatile en-

donuclease involved in nonhomologous DNA

end-joining (47, 61). The protein cleaves

DNA structures with single-strand/double-

strand transitions, such as 3-overhangs, 5-

overhangs, hairpins, flaps, and gaps (49, 50).Artemis interacts with DNA-PKcs, which is

required for its activity. The involvement

of Artemis in specifically processing dam-

aged DNA ends before they can be rejoined

is supported by experiments using Artemis-

deficient cells. In G0/G1 arrested Artemis-

deficient fibroblasts, approximately 10% of

ionizing radiation-induced DSBs remain un-joined for up to 14 days, whereas essentially all

DSBs are rejoined in normal fibroblasts (72).

Three DNA polymerases are implicated innonhomologous DNA end-joining: terminal

deoxynucleotidyl transferase (TdT), and the

translesion DNA polymerases pol mu and pol

lambda (47, 48, 64). Each of these DNA poly-

merases has different properties that will be

useful depending on the structure of the ends

to be repaired. After nuclease processing the

paired ends to be ligated can consist of every

possible permutation of a 3-overhang, a 5-

overhang, and a blunt end, in addition to the

possibility that ligation of both strands is notcoordinated, leading to a small single-strand

gap. While TdT can add untemplated nu-

cleotidestoDNAends,polmuandpollambda

can fill gaps (64, 65). In addition, the transle-

sion DNA polymerases can incorporate nu-

cleotides encoded by the other end of the

break even before the template is covalently

closed.

The core components of nonhomologousDNA end-joining must play a central role in

orchestrating all these activities. They might

serve as a platform bridging the ends to be

rejoined. Ligation could ensue when ends are

compatible and do not require further pro-

cessing. The accessory components required

for the diverse processing reactions may re-

versibly interact with the platform, and theymight be engaged if they can act on the partic-

ular substrate held by the platform provided

by the core components. The central player

of this platform is likely to be the Ku het-

erodimer (Figure 1), which can interact with

Artemis (through DNA-PKcs), pol lambda,

pol mu, and the XRCC4/DNA ligase IV com-

plex (11, 48, 51, 66, 107). Thereversible inter-

action of the processing factors with the coreprovides great flexibility in the combination

of different ends that can be rejoined, because

reversible interaction requires neither a strict

order at which the processing factors engage

northat they actsimultaneously on each of the

two ends or even on each of the two strands

of one end.

WHEN RECOMBINATION ISNEEDED AN ORDERED SERIESOF REACTIONS IS SET IN

MOTION

DSBs are apparently an inevitable conse-

quence of DNA replication (Figure 3).

Such one-ended breaks require homologous



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Joint molecule:invasion of aprocessedsingle-strand DNAtail into ahomologous duplex

leads to a jointmolecule betweenthe broken DNA andits repair template.The invading singlestrand is base pairedwith complementarysequences on onestrand of the duplexin either a theoreticalthree-strandedstructure or with theother strand of the

duplex displacedRecombinase: aprotein capable ofcatalyzing theexchange ofbase-paired partnersbetween two DNAmolecules.Recombinasesinclude RecA frombacteria, RadA(sometimes alsocalled Rad51) from

archaea, and Rad51from eukaryotes.These proteins donot share a highdegree of amino acidsequence similaritybut have very similarstructure and allform similarfilaments bound toDNA

recombination for repair, and, therefore,

homologous recombination is an essential

process. DSB repair through homologous re-

combination is generally accurate because the

undamaged sister chromatid is used as a re-pair template (Figures 2, 3). In order to

re-establish a replication fork, one strand of

the broken end must be base paired with

its complement in the duplex sister chro-

matid through homologous recombination

reactions. Understanding the web of molec-

ular mechanisms that are coordinated to ef-

fect homologous recombination repair usu-

ally starts at the central defining event of theprocess, DNA homology search, and joint

molecule formation where the end to be re-

paired pairs with an intact homologous du-

plex (Figure 2c). This step, at the core of

DSB repair by homologous recombination,

is catalyzed by a recombinase-coated single-

stranded DNA. Our mechanistic understand-

ing is less certain for the steps precedingand following joint molecule formation. As

a first order of business, broken ends need

to be kept in close proximity to their repair

partners. Before pairing with a homologous

partner, broken DNA ends have to be pro-

cessed to create single-stranded DNA with a

3-hydroxyl overhang end. In eukaryotic cells

this is likely coordinated with removal of nu-

cleosomes around the break. In addition, re-combinase function is clearly controlled so

that the recombinase is efficiently loaded onto

breaks that occur during replication, and has

limited activity at other times and places in

the genome. [The obvious exception of ac-

tivating recombination during meiosis is not

considered here (68).] After joint molecule

formation, any DNA sequence information

lost at the break site is recovered by syn-thesis using the intact sister chromatid as a

template and the invading broken end as a

primer. For this to happen, a DNA poly-

merase has to be assembled at this particular

type of primer template structure and initi-

ate synthesis. There are several scenarios for

completing repair after joint molecule forma-

tion that differ for one-ended and two-ended

breaks (Figures 2, 3). Two-ended breaks t

involve joint molecule formation with bo

ends result in double Holliday junctions th

need to be separated into two intact chrmatids (Figure 2dg). Completing repair

one-ended breaks may require reassembl

a replication fork from a joint molecule,

repairing of newly synthesized DNA with

sister chromatid at the original break by p

cesses formally resembling branch migrati

(Figure 3gj). Much of the actual molecu

detail here is theoretical, and what is need

exactly will vary with the type of DNA breand the structure of the recombination pa

ners created to repair it. The required prot

machinery for identifying homology and p

forming strand exchange has been conserv

across the three kingdoms of life and is

viewed elsewhere (16, 91, 113). New inform

tion is emerging on the cellular factors need

to properly coordinate recombination fun

tions for effective DSB repair.

FIRST, KEEP REPAIR PARTNERCLOSE THROUGH ALL THEPRELIMINARY STEPS

When both strands of DNA are sever

the correct repair partners can become se

arated, be it the two ends of a direct bre

or one daughter duplex emanating fromreplication fork. Therefore, an early step

DSB repair is a bandaid procedure to ke

partners close for eventual healing reactio

There are good candidates for these mol

ular bandaids from several organisms, an

mechanistic picture of their molecular fun

tion is emerging. Genetic studies in ye

link both the cohesin complex and the r

lated SMC5/6 complex to DNA repair, sugesting that these proteins, known to be

the business of keeping chromosomes

gether, similarly link broken DNA molecu

to facilitate repair (14, 89). The most mec

anistically advanced concepts for prote

mediated organization of broken DNA

provided by the eukaryotic Rad50/Mre

complex (Figure 4). This complex h

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required functions early in DSB repair when

bandaid action is needed. In cell biological ex-

periments, the Rad50/Mre11 complex is one

of the first factors detected at DSB sites (42). A

role for organizing broken ends in the nucleusis suggestedby thereduced clustering of DNA

ends in Rad50/Mre11 complex-deficient cells

(3). The molecular basis for this can be found

in biochemical and biophysical studies of hu-

man Rad50/Mre11 complex that show it to

form oligomeric complexes on linear DNA

(Figure 4d). Rad50/Mre11 oligomers bound

to DNA work like molecular velcro, through

interactions among the apexes of the 50-nmlong coiled coils of complexes bound to differ-

ent DNA molecules, thereby tethering bro-

ken DNA ends (18, 30, 101). Single-molecule

imaging experiments show that DNA bind-

ing acts as a conformational switch in the

Rad50/Mre11 complex that favors interaction

among coiled-coil apexes of different com-

plexes and thereby promotes DNA tethering(58). The in vivo importance of interaction

between the Rad50 coiled-coil apexes was el-

egantly demonstrated by replacing them with

a ligand inducible dimerization domain. The

rescue of Rad50 function in yeast cells ex-

pressing this variant became ligand depen-

dent, proving the necessity for Rad50 coiled-

coil apex interactions for biological function

(109). Contrary to the usual relationship, themolecular details of keeping brokenends close

are less well developed for bacteria. How-

ever, evidence is emerging that the RecN pro-

tein, structurally related to Rad50, is likely

to have a role in organizing broken DNA

for repair, perhaps in an analogous fashion

(35, 54, 78).

SECOND, CREATE

SINGLE-STRANDED DNA ATTHE BREAK

The next step required for homologous re-

combination repair of a DSB is to process

it into a single-stranded end specifically with

a 3-hydroxyl overhang (Figure 2b). Here

the story is much clearer in bacteria, where

a b c

d e

Figure 4Tethering of broken DNA molecules by the Rad50/Mre11 complex. (a) Thuman Rad50 protein consists of a globular ATPase domain and a 50-nmlong coiled coil. The Rad50/Mre11 complex contains two Rad50 molecudimerized at their globular domains where two Mre11 nuclease subunitsalso located. The stoichiometry of the third subunit of the complex, Nbswhich also resides in the globular domain, is less well defined. (b) The twcoiled-coil arms of the complex are flexible and can interact at their apexthough coordination of a Zn-ion in a so-called Zn-hook structure.Interaction of the coiled coils within a complex prevents its biologicalimportant function, which is to tether broken DNA molecules throughintercomplex interactions. (c) The complex binds DNA (red line) throughglobular domain while the coiled-coil arms protrude away. DNA bindin

induces a parallel conformation of the coiled-coil arms, which prevent thfutile intracomplex interaction while simultaneously promotingintercomplex interactions. (d) On linear DNA the complex will oligomenear DNA ends. (e) Oligomers of the Rad50/Mre11 complex can nowtether DNA molecules to keep them in close proximity before repair.

this is accomplished by the RecBCD heli-

case/nuclease machine, which also concomi-

tantly loads the RecA recombinase (87, 88).Though these functions of RecBCD are long

established, the recent crystal structure of this

complete complex with bound DNA provided

a clear explanation forhow themanyfunctions

of this extraordinary molecular machine are

coordinated. The RecBCD structure shows

that bound DNA ends are split and each sin-

gle strand is threaded into a channel where

one of the two helicase motors pulls on it



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Nucleoproteinfilament: thecomplexes formedbetweenrecombinaseproteins, RecA,

RadA, and Rad51,and DNA. Arecombinase boundto single-strandedDNA initiates strandexchange with ahomologous duplexDNA molecule.Recombinases bindto DNA in thepresence ofnucleotide cofactorand are

DNA-dependentATPases. In a stableform, therecombinaseproteins can bind in aregular right-handedhelical array toDNA, though strandexchange activitylikely requiresdynamicrearrangements

(20, 83). The nuclease responsible for end

processing is located downstream of the he-

licase motors so as to have access to one

strand or the other. Details of how nuclease

access switches from one DNA strand to theother upon encountering a specific sequence,

known as Chi, remain to be elucidated, but

the overall machinery is now understood (86).

In contrast, the equivalent machinery has yet

to be identified in eukaryotic cells. Genetic

evidence in yeast indicates that end process-

ing involves at least the Rad50/Mre11 com-

plex but does not prove that this is the nu-

clease (40). Indeed, the nuclease activities ofMre11 described to date would create a single-

strand end with the incorrect polarity (95);

however, this is also true of RecBCD activity

before modulation by passing a Chi sequence

in DNA (2). Rad50/Mre11 activity may well

be similarly modified by interaction with or

addition of another component. Alternatively,

Rad50/Mre11 may be less directly involvedin end resection but rather act as a neces-

sary cofactor for another nuclease, of which

there are several candidates. The identity of

the componentsresponsiblefor end resection,

the mechanisms controlling their action, and

coordination of this step with the rest of ho-

mologous recombination are important unan-

swered questions in eukaryotic systems. Here

again, there are likely to be several variationsused for the different circumstances in which

DSBs can be created.

Processing a DSB to reveal a stretch of

single-stranded DNA suitable to load a re-

combinase is required for any type of break

to be repaired by homologous recombination.

However, as for nonhomologous DNA end-

joining, the exact structure at the end mayguide specific processing to facilitate eventual

repair that will require using the DNA end

either as a primer for synthesis or as a liga-

tion partner. Thus the DNA end has to be

either a clean 3-hydroxyl or a 5-phosphate.

This means that DNA ends with other chemi-

cal structures have to be specifically processed

or cleaned up. The different requirements for

end trimming and cleaning may be reflected

in differential requirements for specific f

tors in various experimental systems. For

stance, depending on the type of treatme

applied to create DNA breaks, the ends m

need no processing at all (site-specific edonucleases), or may need to be processed

remove covalently bound proteins (topoi

merase inhibitors), or may need trimming

remove chemical groups left when the bac

bone break occurs in the ribose ring (ion

ing radiation). The endonuclease activities

Rad50/Mre11 are suited to play a role he

allowing possible processing of several diffent end structures, and are consistent with

requirement early in DSB repair. SbcCD

bacterial protein complex structurally relat

to Rad50/Mre11 (17), is a potent nucle

that likely processes aberrant DNA structu

in need of repair, including proteins cov

lently attached to DNA ends (12). Identi

ing the eukaryotic factors needed to trim sp

cific end damage will be important becaudifferent end-processing factors might dir

or require different activities in downstre

steps.

THIRD, ASSEMBLINGNUCLEOPROTEIN FILAMENTTHE CORE RECOMBINATION

CATALYSTFor the next step in homologous reco

bination repair, specifically processed DN

ends are coated with a recombinase. T

all-purpose bacterial RecBCD machine loa

the RecA recombinase onto 3-ended sing

stranded DNA as it is produced by

helicase/nuclease. Similarly, the RecFO

proteins collaborate to load RecA onto sing

stranded DNA regions created when damais encountered during replication that nee

to be repaired by homologous recombin

tion (59, 104). In yeast, the Rad52 prote

has an established role early in recombinati

in facilitating recombinasefilament formati

(39). A role analogous to Rad52 in mammal

cells is apparently performed by BRCA2

large body of evidence from cell biolo

372 Wyman Kanaar


11/21

biochemistry, and structural studies indicates

that BRCA2 is involved in controlling the ac-

tivity of the eukaryotic recombinase, Rad51

(23, 81, 82). A specific mechanistic role

for BRCA2 in loading Rad51 onto single-stranded DNA emerged from these studies.

Direct biochemical analysis of human BRCA2

function has been hampered by the inability

to purify this very large protein. However, a

more manageable version of BRCA2, Brh2,

from the fungus Ustilago maydishas been puri-

fied and appears to have the right biochemical

activities. Elegant biochemical experiments

show that Brh2 facilitates loading Rad51 ontoDNA, specifically single-stranded DNA with

thecorrect polarity starting at a double-strand

to single-strand DNA transition that would

be created by resection of a DNA end in

need of repair (117). A pared-down human

BRCA2, including only theDNA-bindingdo-

main and two Rad51-binding domains, BRC3

and BRC4, has in vitro activities similar tothose of Brh2, and further supports a role

for the complete BRCA2 in loading Rad51

onto DNA in need of recombinational repair

(77).

In addition to its possible importance in

producing the single-stranded DNA substrate

for Rad51 binding, the Rad50/Mre11 com-

plex may have a direct, still unproven, role in

loading Rad51. This idea emerges from ex-periments designed to test for chromosome

remodeling at DNA breaks (see below) that

demonstrate a requirement for Rad50/Mre11

to load Rad51 at enzyme-induced specific

breaks, even though these breaks were effi-

ciently processed into single-stranded DNA

(96). It is equally important to keep Rad51

away from sites where recombination is notneeded and may be dangerous (92). The DNA

translocating motor-protein Rad54 can facil-

itate Rad51 filament disassembly in vitro, and

recent in vivo work supports the importance

of this Rad54 function (36, 74, 84, 90, 100,

105). Currently under investigation are the

consequences of Rad51 accumulating where

it is not needed or not disassociating after

strand exchange is complete; these should re-

veal the important biological function of the

recombination mediators involved in control-

ling Rad51 function.

Eukaryotic Rad51 is a problematic recom-binase from the biochemists point of view.

The accumulated in vivo and in vitro data on

theessential natureof Rad51,its functionsand

activities, as well as structural studies led to

the universally accepted view that Rad51 is

the functional homolog of RecA and there-

fore a bona fide recombinase. However, un-

like RecA, Rad51 does not have a strong pref-

erence for binding to single-stranded DNAover double-stranded DNA, and Rad51 is

less active than RecA in ATPase and strand-

exchange assays in vitro (81). This raises sev-

eral questions concerning the nature of active

Rad51 nucleoprotein filaments, the optimal

in vitro conditions, and the possible need for

other proteins in eukaryotic strand exchange

reactions. Different experimental approachesare sorting through these details and provide

some fresh ideas for understanding mechanis-

tic aspects of strand exchange. For instance,

the enigmatic Rad51 paralogs are essential

proteins similar to Rad51 but so far with-

out defined mechanistic roles in homologous

recombination (94). The frequent suggestion

that they areinvolvedin controlling Rad51 fil-

ament function is reasonable and difficult torefute, but is not supported by much data. It

hasatleastnowbeenshownthatRad51Cpref-

erentially localizes to sites of enzyme-induced

DSBs (75). These data, together with the as-

sociation of Rad51 with Rad51C, support a

role for Rad51C in DSB repair in association

with Rad51 filaments, but a role for Rad51C

in controlling Rad51 filament function is stillnot proven.

Contrary to the prominent cartoon view

that the recombinase nucleoprotein filament

is a helical regular static rod, the recombinase

filament is a variable and dynamic structure.

Evidence for filaments with different pitches,

the number of protein monomers per turn,

and the length of their rise along the helix

axis, even within a single filament has longbeen available (23, 44,119).Recent imaging of



12/21

Holliday junction:a mobile junctionbetween two duplexDNA molecules.The point at whichtwo of the strands

between the twoduplexes crossovercan move along theduplexes in a processreferred to as branchmigration

human Rad51 filaments in a manner that did

not require regular structure for analysis and

avoided fixing regularities shows them to be

irregular, especially in the catalytically active

single-strandedDNAbound form (73). Theseimages, as well as single-molecule force spec-

troscopy experiments, suggest dynamic asso-

ciation and disassociation of Rad51 at many

points along DNA molecules that could be

essential for accomplishing strand-exchange

reactions. Molecular insight into the source

of dynamic rearrangements within recombi-

nase filaments comes from atomic resolution

structures of yeast Rad51 and archaeal RadAthat notably reveal ATP binding and hydroly-

sis to occur at the interface of two monomers

(13, 111). There is a wealth of information in-

dicating that thenature of thecofactors bound

at the ATPase site influences Rad51 function

and that this is reflected in filament structure

(44, 73, 113, 119). Several structural features

of Rad51 and its filaments on DNA accountfor these flexible arrangements and, presum-

ably, controlled rearrangements that accom-

pany DNA strand exchange. The ATPase in-

terface in the Rad51 filament has at least

the two alternating arrangements observed in

the crystal structure. The amino-terminal do-

main, which likely binds DNA and contacts

the ATPase core of its neighboring monomer,

is attached to the core by a flexible stretch ofamino acids (13, 119). Thus there are several

hinge points within Rad51 monomers and

between them in a filament that allows for po-

tentially large changes in structure. Perhaps

more important, these multiple flexible con-

nections within the protein polymer and be-

tween the protein and DNA polymers mean

that loss of contact between any two partners,such as at the Rad51 ATPase interface or be-

tween a Rad51 monomer and DNA, does not

result in disassociation of the complex but al-

lows for a variety of dynamic rearrangements

(24, 112). Theextent of these rearrangements,

their control by nucleotide cofactor binding

and hydrolysis, as well as their role in pro-

moting strand exchange are exciting questions

that can now be addressed.

AFTER THE RECOMBINATIONPARTNERS ENGAGE

Most homologous recombination repair

DNA breaks occurs at replication forks whone-ended breaks are produced, as mention

above (15). The DNA structures resulti

from strand invasion and joint molecule fo

mation have crossed DNA strands, calHolliday junctions (Figure 2f, Figure 3

In order to complete repair after recombin

tion, the DNA strands have to be uncross

or cut by structure-specific nucleases cal

Holliday junction resolvases. This is accoplished by the RuvABC complex or the Re

protein in bacteria (106). The search for

equivalent enzyme in eukaryotes has turn

up a complex of two Rad51 paralogs, XRC

and Rad51C, that appears to be associat

with resolvase activity. In addition, extrafrom Rad51C-deficientcells haveno resolv

activity (43). Alternatively, the crossed DNstrands resulting from joint molecule form

tion can be separated by the combined a

tion of a helicase, specifically those in t

RecQ family, and a topoisomerase (28, 11

A true Holliday junction resolvase may n

be needed to complete repair of breaks

recombination at replication forks, and t

exact biochemical activity required to sep

rate the recombined strands will depend where the break originally occurred, for

stance whether leading or lagging strand te

plates were broken. This step in homologo

recombination repair may also require diff

ent mechanisms and enzymes depending

circumstances, similar to the requirement

diverse mechanisms at early steps to proc

different types of broken DNA ends.

BEFORE THE BEGINNING ANDAFTER THE END

So far we have considered advances

understanding mechanistic steps of stran

exchange reactions of homologous recom

nation repair. However, in cells this rep

process is connected to a web of other even

374 Wyman Kanaar


13/21

Exciting advances are also elucidating these

mechanistic connections. Evidence is rapidly

emerging for the role of chromosome remod-

eling as an essential prerequisite to DSB re-

pair, and the molecular components involvedare being identified. Completing repair of

DNA breaks requires the product of recom-

bination, a joint molecule, to become the sub-

strate for replication. New information is also

emerging on this hand-off step and the iden-

tity of the eukaryotic polymerase involved.

BEFORE RECOMBINATION

DSBs are associated with changes in the sur-

rounding chromatin. The dramatic phospho-

rylation of histone variant H2AX was the first

described chromatin alteration that follows a

DNA break. This histone mark may have a

role in activating DNA damage-response cell

cycle checkpoints and recruiting chromatin

remodeling and repair factors (98), althoughthe lack of dramatic DNA repair-defective

phenotype of H2AX-deficient cells and mice

suggests its role might be nonessential (5,

10). Histone acetylation by Trrap-Trip60 also

modulates loading of repair proteins at DNA

breaks (62). Here chromatin immunoprecipi-

tation data indicated that Rad51 association

with enzyme-induced break sites is depen-

dent on Trrap-Trip60. It was suggested thatthe acetylation of histones loosened chro-

matin as a necessary step before homologous

recombination proteins could assemble on

DNA. The chromatin remodeling complexes

INO80 and SWR1 are also specifically impli-

cated in making broken DNA ends accessible

to repair factors (21, 60, 97). The possibility

that certain chromatin changes are associ-

ated with either homologous recombinationor nonhomologous DNA end-joining is still

unclear. In yeast, components of the RSC

chromatin-remodeling complex were identi-

fied in genetic screens for defects in nonho-

mologous DNA end-joining and were also

shown to interact with Mre11, suggesting that

Mre11 recruits RSC to ends where chromatin

remodeling facilitates repair (80). In most of

these cases, the implication is that DNA at

break sites has to be freed of nucleosomes

before repair proteins can have access. Nu-

cleosome removal at HO-induced break siteswas directly demonstrated by chromatin im-

munoprecipitation experiments (96). The loss

of nucleosomes involved the INO80 chro-

matin remodeler and was less efficient if the

Rad50/Mre11 complex was absent.

AFTER STRAND EXCHANGE

Homologousrecombination reactions involv-

ing DNA broken during replication do notcompletely repair collapsed replication forks,

but create DNA structures that can be rescued

by strand-switch synthesis. In this way, one

broken daughter strand will invade the ho-

mologous daughter duplex, creating a primer

with the 3 end of the invading nascent strand

using the other nascent strand as a template

to copy sequence information past the site ofthe break-causing lesion (Figure 3). In or-

der to complete repair, this recombination in-

termediate has to be recognized by a DNA

polymerase as a primer template in a man-

ner coordinated with joint molecule forma-

tion. The accumulated genetic and biochem-

ical evidence provides a clear picture for how

recombination-directed replication can oc-

cur. Joint molecules formed by RecBCD pro-cessing of a double-stranded end, concerted

loading of RecA, and strand invasion yield a

3-hydroxylpairedtoaduplexasaprimertem-

plate terminus. A dynamic RecA filament, ca-

pableofdisassembly,isrequiredatthisstageof

the reaction to expose the DNA for assembly

of replication machinery (115). A complete

replication fork, including the DnaB heli-

case, DnaG primase, andDNApolymerase IIIholoenzyme, can restart from this structure in

reactions that depend on the PriA primosome

(115, 116). Comparable detail on coordinat-

ing replication restart with recombination is

not available for mammalian cells; however,

the translesionpolymerase pol eta participates

in this DNA synthesis reaction both in vivo

and in vitro (34, 53). Colocalization of pol eta



14/21

and Rad51 in DNA damage-induced nuclear

focihadpreviouslybeendescribed(33)butdid

not prove functional interaction or mechanis-

tic connections. The in vitro studies report

direct interaction between pol eta and Rad51as well as Rad51-stimulated pol eta-specific

polymerase activity from a model recombina-

tion intermediate primer template (53).

SUMMING UP AND MOVING ON

Genome stability is normally maintained in

the face of potentially dangerous DSBs by the

combined activity of nonhomologous DNA

end-joining and homologous recombination.

As reviewed here, the reaction that will even-tuallyaccomplish therepair of anygivenDNA

break will depend primarily on whether there

were one or two ends to begin with. Two-ended breaks, not necessarily created with a

sister chromatid in the neighborhood, will be

repaired before they can cause more trouble

by nonhomologous DNA end-joining. One-

ended breaks that occur during replicationwhen DNA polymerases encounter damage or

breaks in the template need homologous re-

combination for repair and are conveniently

associated with the required homologous du-

plex in the form of the other newly synthe-

sized sister chromatid. In addition, the exactchemical structure of the DNA break, and

therefore the manner in which breaks are cre-

ated either in natural or experimental situa-tions, also manipulates the requirement for

processing steps and consequently influences

which factors will affect repair. Therefore,

although categorizing different DSB repair

mechanisms along pathways and subpathways

is conceptually useful, it is unlikely that DSB

repair occurs along ordered pathways because

there is variation in and overlap between therequired molecular machinery.

The general scenario that follows wou

be for components that are needed early

DSB repair to simply associate with the br

ken DNA if they can. Additional compnents would interact reversibly and enga

if their substrates and partners are preseThese combinatorial molecular interactio

are envisioned to stabilize the assembly

molecular machinery responsible for affe

ing eventual repair. Conversely, following i

tial interaction with broken DNA, repair f

tors not stabilized by additional interactio

will disassociate, allowing other possible co

ponents to build up and other mechanisto take over. This suggests that the build-

or stability of functional repair machinery

based at some point on the presence of one

two DNA ends. In simple form, nonhomo

gous DNA end-joining would involve a sta

intermediate with both DNA ends engag

that are then not likely to be stably bou

by homologous recombination proteins t

otherwise recognize ends. Likewise, a sinDNA end, possibly in combination with

duplex partner, would form stable comple

with homologous recombination proteins a

not with nonhomologous DNA end-joini

components.

The molecular details connecting DSB

pair to other genome transactions are now b

coming clear. The exciting coordination btween break-induced chromatin remodeli

and repair reactions is just beginning to

explored. In addition, the molecular collab

rations needed to restart replication follow

recombination at collapsed forks are beg

ning to be identified in mammalian system

We expect that these multiple, connected a

overlapping collaborations will form a w

of molecular interactions from which a pato repair for any number of different DN

breaks can emerge.

SUMMARY POINTS

1. DNA double-strand breaks are potentially very dangerous and vary in structure, andtherefore multiple options for their repair are available.

376 Wyman Kanaar


15/21

2. The structure of the DNA ends themselves influences their path to repair.

3. Two-ended breaks can be repaired by nonhomologous DNA end-joining, which ispossible and active at all stages of cell division cycle.

4. One-ended breaks, created commonly during replication, need to be repaired by

homologous recombination, which is important in the S phase of the cell division

cycle.

5. Variations within the nonhomologous DNA end-joining mechanism have been de-

fined based on the processing of the DSB ends required before joining, and similar

requirements for end processing likely occur in homologous recombination.

6. DNA break-induced chromatin remodeling activities specifically coupled to repair

have been identified, and the role of the remodeling factors in genome stability is

being defined.

7. Important aspects of the molecular level dynamics occurring in the machinery of

homologous recombination, both the early step of keeping ends close and later step

of recombinase nucleoprotein filament, are now being defined by single-molecule

experiments.

8. There is a web of interactions between the molecular components of break recogni-tion, break processing, and break repair that can accommodate a path to repair for

many different broken DNAstructures occurring in many different cellular situations.

ACKNOWLEDGMENTS

Work in the C.W. and R.K. laboratories is supported by grants from the Dutch Cancer Society

(KWF), the Netherlands Organization for Scientific Research (NWO), the Association for

International Cancer Research (AICR) and the European Commission.

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