Chapter 25 DNA Metabolism1. How does a DNA molecule replicate with

high fidelity?

2. How are DNA lesions (damages) repaired to maintain the integrity of genetic i

nformation?

3. How do DNA molecules recombine (rearrange)?

High accuracy, multitude of participants.

1. The deduced double helix structure of DNA revealed the possible ways for its

replication (1953)• Each DNA strand was proposed to act as the templat

e (complement) of the other.• The way a DNA molecule replicates was hypothesiz

ed to be semiconservative: each of the newly synthesized DNA duplexes consists of one strand from the parent DNA and one strand of newly synthesized (Watson and Crick, 1953). (the conservative replication would generate two daughter DNA molecules with one consisting of two new and one of two old strands.)

Old strandNew strand

The hypothesis ofsemiconservativereplication proposed by Watson and Crickin 1953.

2. DNA replication was proved to be semiconservative by the Meselson-Stahl e

xperiment using E. coli cells (1957)• 15N (the Heavy isotope) and 14N (the Light isotope)

was used (as NH4Cl) to label the DNA to distinguish the old and newly synthesized DNA molecules in cells;

• Three types of DNA molecules containing various proportions of 15N and 14N (H-H, H-L, L-L) were separated by centrifugation to equilibrium in a cesium chloride (CsCl) density gradient (only two types would be expected if replication is conservative).

Radioisotope labelingand density gradientcentrifugation clearlydistinguishes replications ofsemiconservative from conservative.

The Meselson-Stahl experiment:DNA molecules duplicate semiconservativelyin E. coli cells.

15N-15N

0 generation

1 generation

2 generations

3 generations4 generations0 and 2 mixed

0 and 4 mixed15N-14N 15N-15N14N-14N

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3. A variety of simple questions were asked about DNA replication

• Are the two parental strands completely unwound before replication begins?

• Does replication begin at random sites or at unique sites? • Does DNA replication proceed in one direction or both

directions? • The overall chain growth occurs in 5` 3`, 3` 5`, or both

directions?• What mechanisms ensure that DNA replicates once per

cell division?

• What enzymes take part in DNA synthesis?

• How does duplication of the long helical duplex occur without the strands becoming tangled? …...

4. Autoradiography studies: daughter strands are synthesized immediately after p

arental strands separate • Electron micrographs of the autoradiographs of replicating

plasmid, SV40 virus, and E. coli chromosomal DNA with 3

H-thymidine incorporated revealed -like structures: no single stranded DNA was visible.

• The chromosomal DNA of E. coli is a single huge circle!• No temporary creation of linear DNA occurred during replic

ation of the circular DNA.

A electron micrograph of the replicationintermediate of a plasmid DNA: -shaped structures were observed; no single strandedDNA is visible.

No complete unwinding of the two parental strands occurred before the daughter strands are synthesized

ReplicatingSV40 DNA

Unreplicated, positive supercoilsof parent strands

Replicated DNA

No complete unwinding of chains!

Autoradiogram of a replicating E.colichromosomal DNAlabeled by [3H]thymidine.

No complete unwinding of DNA chains!

5. DNA replication was found to begin at specific sites and proceed bidirectionally

• Pulse-chase labeling studies of replicating DNA, as well as direct EM examination of intermediates of replicating linear T7 bacteriophage DNA all revealed that DNA replication is bidirectional.

• Denaturation mapping studies with a series of replication intermediates of circular DNA and direct observation of a linear DNA revealed that DNA replication begins at specific replication origins.

Autoradiogram of replicating mammalian cellular DNA pulse-chase labeled with [3H]thymine: replication is bidirectional.

Examination of T7 DNA (linear) replication using electron microscopy:1. The daughter polynucleotide strands are synthesized almost as soon as the parental strands separate (no complete unwinding of chains);2. Replication always began at a specific internal site (not from the ends, not random);3. The replication proceeds in both directions (determined by measuring the distance between the replication fork and the ends)

Replicationorigin

Replicationforks

Denatured loops(single-stranded)

Nondenatured DNA(double-stranded)

Denaturation mapping: the denatured loops are reproducible and thus can be used as points of reference (DNA replication starts at specific origins).

6. The chemistry of DNA polymerization was revealed by in vitro studies using a D

NA polymerase purified from E. coli• Arthur Kornberg purified DNA polymerase I, a mono

meric 103 kDa protein, from E. coli in 1955 and revealed the major features of the DNA synthesis process.

• DNA polymerase I was found to catalyze DNA polymerization in vitro in the presence of a single-stranded DNA template, a preexisting primer with a free 3`-OH group and the dNTPs (using radioactive precursor nucleotides to label and trichloroacetic acid to precipitate the newly synthesized DNA).

• The fundamental reaction for DNA synthesis is a nucleophilic attack by the 3`-OH group of the growing strand on the 5`--phosphorus of a incoming dNTP selected via base-pairing: the newly synthesized DNA is always extended in the 5` to 3` direction.

7. DNA polymerase I is a 103-kDa trifunctional protein having one polymerase and t

wo exonuclease activities• The 3` 5` exonuclease activity was found to be able to re

move mismatched base pairs, thus to proofread the newly incorporated nucleotides (increasing the accuracy by 100 to 1000 fold).

• A “sliding back” model was proposed for DNA polymerase I to proofread mismatched base pairs.

• The enzyme can be cleaved into two parts by mild protease treatment: the small fragment contains the 5’ to 3’ exonuclease activity and the large (called the Klenow fragment) contains the rest two activities.

• The 5` to 3` exonuclease activity is unique for DNA polymerase I, enabling it to catalyze the nick translation process: an RNA or DNA strand paired to a DNA template is simultaneously degraded and replaced; an activity used for both DNA repair and the removal of RNA primers in DNA replication (clean-up functions), also for incorporating radioisotope-labeled dNTPs into a DNA probe (in vitro labeling).

DNA polymease I is proposed to slide backto proofread a mismatchedbase pair using its 3` to 5`exonuclease activity.

DNA polymerase I has three enzymatic activities in a single polypeptide chain, which can be cleaved into two functional parts by mild protease treatment.

Protease cleavage

68 kDa35 kDa

The 3` to 5` exonucleasedomain

The polymerasedomain

Structure of the Klenow fragmentof DNA polymease I

The 5` to 3` exonucleaseand polymerase activitiesof DNA polymerase I allows the enzyme to catalyze the nick translation process of a DNA or RNA fragment base paired with a template.

32P-dATP

Arthur Kornberg wonthe 1959 Nobel Prizein Medicine for hisdiscovery of the mechanism in the biological synthesis of deoxyribonucleic acid (before Watson and Crick won theirs!)

8. The synthesis of the two daughter DNA strands was found to be semidiscontin

uous

• At a replication fork, the overall elongation direction for one daughter strand is 5` to 3` and 3` to 5` for the other due to the antiparallel features of DNA duplexes.

• DNA polymerase catalyzing 3` to 5` extension was hypothesized but never identified.

• Reiji Okazaki discovered ( in the 1960s) that a significant proportion of newly synthesized DNA exists as small fragments!

• These so-called Okazaki fragments was found to join together by DNA ligases to form one of the daughter strands;

• Thus both daughter strands are synthesized in 5` to 3` direction.

• One daughter strand at the replication fork is synthesized continuously and the other discontinuously, called the leading and lagging strands respectively.

Overall direction of progeny chain growthat a replicating fork: one in 5’ 3’ and the other in 3’ 5’ direction.

Both daughter strands at the replication fork are synthesized in5’ 3’ direction, but one (the leading strand) is synthesized continuously and the other (the lagging strand) discontinuously(synthesized initially as Okazaki fragments).

The leading strand

The lagging strand

9. DNA polymerase I was found to be not responsible for DNA replication in E. c

oli cells• The reaction velocity of this enzyme is too low to account fo

r the observed rates of fork movement in vivo.• Its processivity—the average number of nucleotides added be

fore it dissociate from the template-- is too low (about 50 nucleotides).

• E.coli cells having a defective DNA polymerase I were found to be still viable, although sensitive to UV light (1969).

• Two more DNA polymerases (II and III) were discovered in E.coli cells in the early 1970s (~15 years after Pol I was discovered!) and two more in 1999.

10. DNA polymerase III is responsible for DNA replication in E.coli cells

• DNA polymerase III is a multimeric enzyme complex containing at least 10 different subunits.

• The holoenzyme seems to exist as an asymmetric dimer (one is believed to be used for synthesizing the leading and the other the lagging strand).

• The polymerization and proofreading activities are located on separate subunits, and respectively (the enzyme has no 5` to 3` exonuclease activity).

• The dimer provides high processivity, with more than 500,000 nucleotides added per binding.

• The rate of DNA synthesis is high, with 1000 nucleotides/second (only about 20 for Pol I).

Sliding clamp subunits

Catalyticsubunit

Catalyticsubunit

3’ 5’ exonucleasesubunits

Clamp loader

Proposed architecture of DNA polymerase III holoenzyme: an asymmetric dimer

No 5` to 3` exonuclease activity

DNA duplex(infered)

A clamping dimer (determined)

The two subunits ofE. coli polymerase IIIform a circular clampthat may surroundDNA to increase itsprocessivity.

11. DNA polymerase III and many other proteins are part of the replisome for DN

A replication in E.coli cells• In vitro studies revealed about 20 proteins are involved in DNA

replication in E.coli cells.• These include:

– the helicase (dnaB) for moving along the DNA and separating (unwinding) the two DNA strands using energy from ATP;

– the topoisomerases for relieving topological (torsional) strains in the helical structure (positive supercoils) generated during strand separation;

– the Single-stranded DNA-binding protein (SSB) for binding and stabilizing separated DNA strands;

– the primase (dnaG) for generating a short RNA primer;

– The multimeric DNA polymerase III for polymerizing and proofreading the nucleotides according to the templates;

– the DNA polymerase I (polA) for removing the RNA primers and replacing it by a DNA sequence;

– the DNA ligase for sealing nicks.

12. A 245 bp fragment in the E.coli chromosomal DNA, OriC, was identified to be

the replication origin • It was identified by using origin-lacking plasmids.• OriC contains a tandem array of three 13-mer with nearl

y identical sequences and four 9-mer repeats, all of which highly conserved among all bacterial replication origins.

• OriC contains 9 GATC (palindromic) sequences, which can be methylated at the base ring of A, which is believed to be important in regulating the frequency of DNA replication: having only one replication per cell division.

OriC, the replication origin of E. coli chromosome, contains three repeatsof 13 bp ad four repeats of 9 bp.

GATC 5GATC

13. The molecular details of the replication of E. coli chromosomal DNA are the bes

t understood by in vitro studies • The whole process can be divided into three stages:

initiation, elongation, and termination.• During the initiation stage, the DNA is open at the O

riC site and a prepriming complex is formed, with the participation of at least nine proteins.

• During the elongation stage, short RNA primers are first synthesized by the primase, which is then extended by the DNA polymerase III, and finally ligated by the DNA ligase.

• During the termination stage, the two replication forks meet at the Ter sequences, the replisome dissociates, and the two catenated chromosomes are separated by DNA topoisomerase IV.

14. Multiple proteins participate at the initiation stage of DNA replication of the E.

coli chromosome• The replication process begins when a single complex of abou

t 20 DnaA protein molecules (with ATP bound) binds to the four 9 bp repeats at OriC that is (negatively) supercoiled.

• Aided by HU (a histone-like protein), the bound DnaA complex opens the two DNA strands at the AT-rich 13-mer repeats.

• DnaB, the helicase, binds (aided by DnaC protein) to the opened DNA strands as two hexamer clamps and further opens (unwinds) the strands in both directions, thus forming the prepriprepriming complexming complex.

The prepriming complex is formed at the OriC, with theparticipation of DnaA, HU,DnaB, DnaC and otherproteins.

The preprimingcomplex

15. The leading strand is synthesized continuously but the lagging strand disconti

nuosuly • Further unwinding of the DNA duplex needs DNA gyrase (

a topoisomerase II) to relieve the (positive) supercoils generated ahead the replication forks and SSB for stabilizing the unwound single strand DNAs.

• RNA synthesis was found to be needed for DNA replication.

• Kornberg discovered that the nascent DNA is always covalently linked to a short stretch of RNA.

• The RNA primers were found to be 10-60 nucleotides in length.

• The primase (a RNA polymerase), recruited to the open templates via DnaB (the helicase), was found to be the enzyme that catalyzes the synthesis of the RNA primers.

• For the leading strand, only one primer is synthesized, which is then elongated by DNA polymerase III in a continuous way.

• On the lagging strand, DnaB intermittently recruits DnaG (the primase) to form a complex called primosome (引发体 ), and repeatedly making primers for the Okazaki fragments.

• Each Okazaki fragment is then synthesized on one RNA primer by DNA polymerase III.

• The RNA primers are removed and replaced by a DNA sequence in a reaction catalyzed by DNA polymerase I, using its nick translation activity (5` to 3` exonuclease + polymerase).

• The final Okazaki fragments are joined together by the DNA ligase.

• DNA ligase catalyzes the formation of a phosphodiester bond between a 3`-OH group and a 5` phosphate group at a nicked DNA duplex, where the 5` phosphate is first activated by being modified by an AMP group (adenylylation) coming from NAD+ (for the bacterial enzyme ligase) or ATP (for the virus and animal ligases).

Only one RNA primer is needed for synthesizing the leading strand.

(DnaB)

RNA primers arerepeatedly formedby the primase on the lagging strand.

DNA polymerase I replaces the RNA primers byDNA sequences and DNA ligase seals the nicks

DNA ligase first transfersan AMP moiety to the 5`phosphate group (from NAD+ or ATP) beforemaking a phosphodiesterbond.

16. The leading and lagging strands are believed to be synthesized coordinately by a single asymmetric DNA polymerase III di

mer• The two polymerase III cores (having the subu

nits) are believed to be connected by two subunits.• The coupling is believed to accomplish via looping

of the lagging strand template.• The Pol III on the lagging strand intermittently relea

ses the sliding clamp and the completed Okazaki fragment before rebinding to a new subunit dimer loaded at a RNA primer and then synthesizing a new Okazaki fragment.

• Accumulating evidence seem to support that the Pol III dimer complex is associated with the plasma membrane and does not actually itself move; the DNA moves through the fixed complex.

Sliding clamp subunits

Catalyticsubunit

Catalyticsubunit

3’ 5’ exonucleasesubunits

Clamp loader

Proposed architecture of DNA polymerase III holoenzyme: an asymmetric dimer

No 5` to 3` exonuclease activity

17. E.coli DNA replication ends at a specific terminus region with multiple copies

of a 20 bp Ter sequence• The Ter sequences are positioned in two clusters with

opposite orientations.• The Tus protein can bind to the Ter sequences.• At each round of DNA replication, only one Tus-Ter c

omplex seem to function to arrest a replication fork from one direction, with the opposing replication fork stopping when the two collide.

• The Ter sequences do not seem to be essential for stopping replication.

• The two newly synthesized circular chromosomal DNAs are topologically interlinked (catenated) and is finally separated by the action of a Type II topoisomerases .

Replication terminators may help halt the replicationforks, but seems to be not essential for stopping DNAreplication in E. coli.

The newly synthesized two chromosomalDNA molecules areinterlinked (catenated)And are separated byDNA topoisomerase IV.

18. Methylation of GATC sequences at oriC is believed to control the replication fr

equencies in E. coli cells• Hemimethylated oriC was found unable to initiate another round

of DNA replication.• It is believed that the hemimethylated GATC sequences at OriC is

sequestered in the plasma membrane immediately after one round of DNA replication, thus blocking another round of replication until the GATC sequences are released and fully methylated by Dam (DNA adenine methylation) methylase.

• The delay of methylation is hypothesized to limit DNA replication to occur once per cell division.

• The slow hydrolysis of ATP by DnaA protein was also proposed to regulate replication initiation.

Hemimethylatd OriCseems to be sequensteredin the plasma membrane,and thus being not active for initiating DNA replication

Active Inactive

19. DNA replication in eukaryotic cells use essentially the same principles but bei

ng more complex in the details• The best understood eukaryotic replication system is that

of SV40 and yeast.• Formation of the RNA primers and the initial incorporati

on of deoxynucleotides are believed to be catalyzed by DNA polymerase (which has no proofreading activity).

• Later chain extension is believed to be catalyzed by DNA polymerase, which has proofreading activity and is clamped onto the DNA templates via the PCNA trimer rings.

• Specific sequences (~150 bp) that can function as replication origins have only been identified in yeast ( is called autonomously replicating sequences, or ARS) and SV40 virus.

• The rate of DNA synthesis in eukaryotic cells is about one tenth of that of the E.coli cells.

• Replication in the eukaryotic genomes begin at many replication origins.

• 9 purified proteins are needed to replicate SV40 virus DNA in vitro.• T antigen--a multifunctional site-specific DNA binding protein enco

ded by SV40 DNA, binds to the origin (as DnaA) and melts the duplex DNA (as DnaB);

• RPA– encoded by the host mammalian cells and binds to the melted single-stranded DNA (as SSB).

• Pol /primase-- synthesizes the RNA primers and a stretch of DNA sequences, has no proofreading activity.

• Pol – replaces Pol /primase to further extend the RNA-DNA strand, has proofreading activity.

• PCNA (proliferating cell nuclear antigen)-- a trimeric ring-shaped protein that clamps Pol onto the DNA template.

• RFC (replication factor C)-- a clamp loader for PCNA. Topoisomerases-- Relieves the torsional strain induced by the growing replication fork.

• Ligases--joins the Okazaki fragments (which is much shorter than those in E.coli cells), as well as the leading strand.

Model of in vitro replicationof SV40 DNA by eukaryotic enzymes:1. T antigen (Tag) binds and unwinds replication origin;2. RPA (or RFA) binds to single-stranded DNA;3. Pol -primase synthesizes the primers (RNA + DNA).4. RFC loads PCNA to the template.

5. PCNA displaces Pol-primase and functions as a DNA clamp;6. Pol replaces Pol-primase and further extends the DNA strands.

20. Maintaining the integrity of the genomic DNA is essential to all cells

• The DNA molecules can become damaged via a variety of internal (e.g., autonomous deamination of bases) or external (e.g., exposure to UV light and chemical agents) processes.

• A diversity of repair systems have evolved : mismatch repair, base-excision repair, nucleotide-excision repair and direct repair are the common types found.

• Many DNA repair systems are energetically expensive (very inefficient comparing with other metabolic pathways) and redundant (especially to some common types of lesions).

• Unrepaired DNA damages can introduce mutations, which can

cause cancers in mammals (e.g., xeroderma pigmentosum, 着色性干皮病 , is caused by a defect of the DNA repair systems).

• Most carcinogens are found to be strong mutagens: the Ames Test has been used to examine whether a chemical is a potential human carcinogen by testing its mutagenicity on certain bacterial strains.

• Complementary synthesis (based on the duplex DNA structure) after elimination of the damaged nucleotides is a common principle for all the repair systems.

Ames Test:carcinogens aretested for theirmutagenicity onbacterial genomes.

Plating ofhis- salmonellatyphimurium

High level of mutagen

Medium level of mutagen Low level of mutagen

No mutagen Back mutations

21. The mismatch repair pathway acts to increase the fidelity of DNA replication

• In vitro studies showed that the transient undermethylation of GATC sequences in the newly synthesized strand permits strand discrimination in E.coli (not in eukaryotic cells): mismatches are corrected according to information provided by the parent strand.

• At least 12 proteins are involved in mismatch repair in E. coli, among which the MutS-MutL complex acts to recognize all the mismatches (except C-C) and MutH acts to recognized GATC sequences and generates a nick on the 5` side of the G in the (5`) GATC in the newly synthesized (unmethylated) strand when the two meet.

• Exonucleases (I or X cleaves from 3` to 5`direction; VII or RecJ cleaves from 5` to 3` direction) remove a segment of DNA from the newly synthesized strand including the mispaired nucleotide.

• DNA pol III resynthesizes the removed segment.

• DNA ligase reseals the gap on the newly synthesized DNA.

The Mut L, MutS, and MutH proteins in E. coli recognize the mismatched base pairsand generate a nick at a GATC sequence up to 1kbaway.

MutL links MutS (recognizing the mismatch and MutH (recognizingthe GATC sequence)

MutS

A proposed model for methyl-directed mismatch repair in E. coli cells.

Mismatch on the 3` sideof the cleavage site

Mismatch on the 5` side of the cleavage site

22. Base-excision repair acts by removing the damaged bases first

• The key enzymes for this class of repair include the specific DNA glycosylases (e.g., uracil, hypoxanthine, 3-methyladenine, 7-methylguanine, and pyrimidine dimers glycosylases) and the nonspecific AP (apurinic or apyrimidinic) endonucleases.

• The DNA glycosylases remove specific DNA lesions by cleaving the N-glycosyl bonds.

• AP endonucleases cleave the phosphodiester bond near the AP site (either on the 5` or the 3` side of AP).

• DNA polymerase I replaces a segment of DNA including the AP site and DNA ligase seals the gap.

The proposed modelfor base-excisionrepair using specificglycosylases and nonspecific AP endonucleases.

23. Nucleotide-excision repair acts by removing a short fragment of ssDNA conta

ining the lesion• Usually works when the DNA lesion (e.g., the presence of c

yclobutane pyrimidine dimers or 6-4 photoproducts) causes large distortion in the helical structure and is probably the most important way for DNA repair in cells.

• in E. coli, ABC exinuclease, a complex of three proteins (UvrA, UvrB, and UvrC), scans the lesion (A2B), generates two cuts (3` side by B and 5` side by C) on the two sides of the damaged site.

• A 12 mer segment of DNA spanning the damaged bases is released by the UvrD helicase.

• DNA Pol I fills the gap and DNA ligase seals it.

• The exinuclease in eukaryotic cells contains 16 polypeptides, with none homologous to that of the E.coli enzyme complex.

• A 29 mer is released by the human exinuclease.

24. Direct repair acts by directly reverse the base modifications without removing

a base or nucleotide

• Pyrimidine dimers can be directly converted to two free monomeric pyrimines in a reaction catalyzed by DNA Photolyases via free radical intermediates.

• The methyl group of O6-methylguanine (introduced by alkylating agents) is directly transferred to a Cys residue on O6-methylguanine-DNA methyltransferase (not an enzyme in strict sense).

• The methylated O6-methylguanine-DNA methyltransferase act as a transcription activator for its own gene and a few other genes also encoding repair enzymes.

A proposed actionmechanism for thephotolyases

Chromophores

Three unstable radicals

O6-methylguanine-DNAmethyltransferase

Cys-S CH3

O6 methylguanine on DNA isdirectely repaired by removingthe methyl group by a Cys residue of the methyltranseferase

A transcriptionactivator

25 Recombinational repair or error-prone repair have to occur when the complementary strand is also damaged or absent

• These types of lesions include double-strand breaks, double strand cross-links, both complementary strand are damaged or one is damaged with the other absent (as will occur when a replication fork encounters an unrepaired lesiono r strand break).

• The lesions can be repaired by using information from a separate, homologous chromosome via recombinational DNA repair (discussed later).

• When the DNA strands are extensively damaged, the SOS response and error-prone repair (or translesion replication, being a desperate strategy) will occur.

• Some proteins may act to repair lesions (the ABC exinuclease, RecA, Pol II), some to inhibit cell divisions (e.g., sulA), and others to act in translesion replication (Rec A protein, SSB, DNA polymerase V made of UmuC and the shortened UmuD or DNA polymerase IV encoded by the dinB gene).

• Random nucleotides may be added in place of the damaged ones during translesion replications, but the detailed mechanism has not been revealed.

• The increased mutations may generate a few cells (although almost all dead) that are better fit to survive the catastrophic conditions (better species may thus evolve).

When a replication fork passes the unrepaired lesions situations will be generated where both complementarystrands of a duplex are unable to act as a template.

26. DNA rearranges in cells via genetic recombination

• Blocks of genes from homologous chromosomes (e.g., between homologous paternal and maternal chromosomes) were found to be exchanged by the process of crossing over, or homologous recombination during Drosophila meiosis (early 20th century).

• Homologous recombination was then found to occur commonly in all types of organisms between two DNA sequences sharing homology.

• Two more major types of DNA rearrangements were later revealed.

• Site-specific recombination was found to occur when the DNA of phage integrates into the E.coli genome, where the DNA exchange occurs only between specific DNA sequences.

• Site-specific recombination was also found to occur in rearranging certain genes in all types of eukaryotic cells.

• Certain DNA elements, called transposons, were found to be able to randomly transpose (mobilize, hop) from one location to another on chromosomes, thus called DNA transposition (speculated by Barbara McClintock while studying maize genetics in the 1950s), which was also found in bacteria and animals many years later.

• All the strands of the recombining DNA molecules have to be broken and rejoined with new strands for all these recombinations.

• Novel DNA intermediates with unusual structures (with three or four strands interwound) have to be formed.

• Principles of the three kinds of recombinations have been widely used to carry out artificial manipulations of DNAs (or genetic engineering) in all types of cells.

Homologous recombination is believed to occur between the closely associated chromatids (in tetrads)during the prophase of the first meiotic division of germ-line cells to produce haploid gametes.

Current studies show that these observed“crossover” sites may not promotebut inhibit DNA exchange.

EM examination of a chiasmata: no contactbetween homologous DNA molecules!

27. Homologous DNA recombinations during meiosis is believed to be initiated wit

h double-strand breaks• The two homologous DNA molecules, with one hav

ing a double-stranded break, have to be first aligned.• Two 3` single strand extensions are then generated b

y the action of an exonuclease.• Two crossover structures called Holliday intermedi

ates are then formed by the invasion of the exposed 3` ends in the intact duplex DNA followed by branch migration and replication, resulting in a region of heteroduplex DNA.

• Four (two for each duplex) cleavages of the Hollida

y intermediates followed by rejoining result in either two nonrecombinant or recombinant DNA duplexes.

• Holliday intermediate was directly observed between plasmid and virus DNA molecules.

• Such homologous recombination leads to the generation of genetic diversity, orderly segregation of chromosomes, or repair of several types of DNA damages.

Isomeric Holliday intermediates between two plasmids

Single-strandedring

28. Proteins involved in homologous recombination have been identified in E.coli

• RecBCD enzyme (encoded by three genes): – Able to bind to a free blunt end of a DNA (double

strand breaks) and travel along the duplex with its helicase activity unwinding the duplex, meanwhile degrading both single strands using its dual 5’ 3’ and 3’ 5’ exonuclease activities.

– when RecBCD encounters a chi sequence, it generates a 3` single-stranded extensions (A total of 1009 chi sequences have been revealed in E. coli).

• RecA protein: able to form nucleoprotein filament on single-stranded

DNA or duplex DNA with a single-stranded gap in vitro; able to promote strand exchange, probably via a spooling action, between three or four homologous DNA strands in vitro; is believed to mediate strand exchange and branch migration to form the Holliday intermediate in vivo.

• RuvC, the resolvase, cleaves the Holliday intermediate to generate recombinant or nonrecombinant products.

• Topoisomerases, DNA polymerases and DNA ligases also act during DNA recombination.

• Much more studies are needed to fully understand homologous recombination.

The RecBCD enzyme binds toa double stran DNA break,cleaves both strands, generatesa 3` single strand extension When meeting a chi sequence with a free –OH group.

5` GCTGGTGG 3`

EM picture and computer imagingmodel of anucleoprotein filamentformed between RecAand a single-stranded DNA.

29. DNA lesions unrepaired will stall the replication process and are believed to be repaired via homologous recombination

before replication restarts • A lesion in a single-strand gap is repaired via a proc

ess requiring RecA, RecF, RecO, and RecR.• A double strand break is repaired via a process requi

ring RecA and RecBCD.• After fixation, replication restarts using the replicati

on restart primosome.• All three aspects of DNA metabolism work together

to repair halted replication forks!

Replication forks stall at unrepaired lesions, which is then fixed via homologous recombination before replication restarts via the replication restart primosome.

30. Site-specific recombinations occur only between specific DNA sequences of 20-200bp and are catalyzed by specific reco

mbinases

• Site-specific recombinations have specialized functions (including virus integration into host genomes; regulation of expression of certain genes; programmed DNA rearrangements during cell differentiation).

• In vitro studies of different site-specific recombination systems seem to suggest a common reaction pathway:

• First a specific (tetrameric) recombinase binds to two specific DNA sites with the short nonpalindromic sequences aligned in the same orientation.

• Short stretches of identical sequences are usually shared by the two sites.• One DNA strand in each DNA duplex is cleaved at a specific point, for

ming two transient covalent intermediates between the DNA and the recombinase (similar to what happens when topoisomerases act).

• The two free ends at the cleaved site are then exchanged and joined to a new partner, generating a Holliday intermediate.

• An isomerization of the Holliday intermediate switches the position of the newly joined strands and the yet-cut strands (accomplished by a rotation of the recombinase).

• Another round of chain breakage and rejoining complet

es the whole recombination process.• In some systems, both strands of each recombining DN

A segment are cut concurrently and rejoined to new partners (Holliday intermediate would not be formed in these cases).

• The chains exchange reciprocally and precisely during site-specific recombination.

• The recombinase acts as site-specific endonuclease, as well as DNA ligase.

31. A site-specific recombination leads to DNA inversion, deletion or insertion

depending on the location and orientation of the recombining sites

• When the two recombination sites are located on the same DNA molecule, an opposite orientation leads to inversion of the intervening DNA fragment; a common orientation leads to deletion.

• When the two recombination sites are located on two different DNA molecules, insertion will be generated if one or two of the DNA molecules are circular.

Site-specific recombination on the same DNA molecule leads to either inversion or deletion ofthe intervening DNA fragments.

32. Bacteriophage DNA can be integrated into and excised from E.coli genomic

DNA via site-specific recombination

• This is the first site-specific recombination system identified and studied in vitro.

• The integration is accomplished by a phage-encoded recombinase called the integrase, where exchange of single strands probably take place sequentially.

• Excision of the phage DNA from the bacterial genomic DNA needs auxiliary proteins other than the recombinase.

Integration of bacteriophage DNA into E.coli genomic DNA viasite-specific recombination1. The recombinase ( integrase) is encoded by the phage DNA;2. The recombination sites (attP on the phage DNA, attB on the bacterial DNA) share 15 bp of complete homology;3. Excision of the DNA from the host genomic DNA occurs when the host cells are under stress conditions, using different recombining sites and different auxiliary proteins (FIS and XIS, encoded by the bacterium and phage respectively.

15 bp

33. The large number of different antibodies are generated via site-specific reco

mbinations in vertebrates• A human can generate about 108 different antibody proteins,

each having different binding specificities.• The whole human genome consists of about 4 X104 genes.• This antibody diversity was found (in 1976 by Tonegawa) t

o be generated from site-specific recombination during the development of antibody-generating mature B cells from stem cells.

• Segments of DNA are randomly joined together via site-specific recombination to form the millions of genes encoding the light and heavy chains of the 108 different antibodies.

• RAG (recombination activating gene) proteins catalyzes the formation of double strand breaks and hairpin ends between the recombination signal sequences (RSS) and the V or J segment.

• The V and J segements are then joined (with variations) by the action of a second complex of proteins.

Programmed DNAdeletions resulted from site-specific recombinationslead to the diversityof immunoglobulins.

A proposed mechanismon the removal of the intervening DNA betweena V and a J segment

34. Transposable genetic elements move from one location to another via

• DNA transposition was originally speculated by Barbara McClintock (1940s) while studying maize genetics, later found to be present in all types of cells;

• DNA transposition occurs randomly, but very infrequently.

• The transposable genetic elements can be divided into two families, transposons move directly as a DNA fragment and retrotransposons move via a RNA intermediate, sharing similarity to retroviruses.

The two major classes of mobileDNA elements

35. DNA transposition in bacteria are either direct or replicative

• There are two classes of transposons in bacteria • Insertion sequences, being the simple transposons contai

ning only the sequences required for their transposition and the genes for transposases that promote the processes.

• Complex transposons, contain additional genes that often code for proteins confer resistance to antibiotics (being one source of generating drug-resistance bacteria).

• Most bacterial transposons have short repeats at the two ends, serving as binding sites for the transposases.

• A short sequence at the target site (5-10 bp) is al

ways duplicated (flanking each end of the inserted transposons), reflecting the cutting mechanism used to insert a transposon into the target site.

• DNA transposition in bacteria can be either direct (leaving a double strand at the donor DNA) or replicative (leaving the donor transposon intact)

The staggered cuts generated by the transposases at the target sites lead to the duplication of shorttarget sequences at the two ends of the inserted transposons.

DNA transposition in bacteria can be eitherdirect or replicative.

Summary• DNA replication begins at specific origins, is semiconserva

tive, bidirectional, and semidiscontinuous.• All DNA polymerases need a primer, extend the DNA chai

n in 5` to 3` direction (using dNTPs), and have 3` to 5` exonuclease activity for proofreading.

• DNA polymerase III in E. coli, DNA polymerase and in eukayrotic cells are responsible for DNA relication in vivo.

• DNA replisomes contain many protein components including helicases, single-stranded DNA binding proteins, topoisomerases, primases, ligases etc.

• It is likely that one DNA polymerase complex catalyzes the synthesis of both the leading and lagging strands at each replication fork via DNA looping.

• Complex and redundant DNA repair systems have evolved to correct lesions in the DNA molecules.

• The multiple DNA repair systems include mismatch repair (using Mut L, MutS, and MutH proteins in E.coli), base-excision repair (using specific DNA glycosylases and nonspecific AP endonucleases in E. coli), nucleotide-excision repair (using a excinuclease complex), direct repair (using specific enzymes or other proteins), homologous repair, and error-prone repair (use under desperate conditions).

• DNA recombinations include different types: homologous, site-specific, and transposition.

• Homologous recombination occurs between any two homologous sequences, and is believed to be initiated by double-strand breaks, with RecBCD (helping to generate a 3`-terminal single-stranded end near the chi sequences), RecA (act to promote strand exchange and branch migration), RuvC (to resolve the Holliday structure) actively involved in E. coli.

• Site-specific recombinations occur between DNA molecules (or regions) having specific sequences and is catalyzed by specific recombinases.

• Transposable genetic elements, found in all types of organisms, jumps from one chromosomal location to another via either direct transposition or replicative transposition.