DNA replication and repair - Lecture 3

Jim Borowiec

September 28, 2006

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Overview of DNA replication

TelomereTelomere Centromere

DNA chromosome

Specialized elements termed'origins of DNA replication’

occur many times on achromosome

Origin of DNA replication

Initiation of DNA replicationfrom origin of replicationgenerates structures termed'DNA replication bubbles'

End replication problem

DNA replication(from internal regions of the chromosome)

3’

5’

3’

5’

by leading strand synthesis

+3’

5’

by lagging strand synthesis

iDNA

RNA primer

Processing

3’

5’

DNA

replication

3’

5’

loss of DNA

After multiple rounds of DNA replication, genetic information will be lost

Replication of telomeres

•Telomeres contain many copies of a specific DNA repeat

(TTAGGG)nTTAGGGTTAGGG-3’(AATCCC)nAATCCC-5’

•Involves special RNA-containing polymerase called telomerase

TelomeraseCCAAUCCC

RNA template

5’

•Telomerase adds one or more copies of the TTAGGG repeat, preventing DNA loss

(TTAGGG)nTTAGGGTTAGGGTTAGGG-3’(AATCCC)nAATCCC CCAAUCCC

5’

Senescence

‘Hayflick limit’

Somatic cells

‘Hayflick limit’

Widespread cell death

Crisis

Germ line cells

Senescence

p53 mutation

Somatic cells

‘Hayflick limit’

Widespread cell death

Crisis

Germ line cells

Senescence

p53 mutation

Telomerase activation

Telomere stabilization

Somatic cells

Telomere length

Cell Divisions

Germ line cells

Telomerase needed for cell immortalization

Most somatic cells do not have telomerase activity

Mechanisms to repair damaged DNAor mispaired DNA

Usually involves synthesis of portions of only one DNA strand

Involves synthesis of 1 to >1000 nt depending on type of repair reaction

Types of DNA damage

1. Spontaneous

C. Oxidative damage to bases (life span of an organism is inversely correlated with metabolic rate/DNA oxidation)

B. Loss of bases - depurination and depyrimidation (~5000 purines are lost per human cell per day)

A. Base deamination (ex: cytosine is converted to uracil at a rate of ~100 bases per human cell per day)

2. Environmental damage

B. Chemical agents (e.g., benzo[a]pyrene)

A. Radiation (ionizing and ultraviolet)

Surveillance

•For all types of DNA damage

•Surveillance factors with different recognition specificities continually scanning the DNA for damage or mispairs

•Upon finding a damage or mistakes, surveillance factors recruit other repair factors

Signal to recruitadditional repair

factors

sugar-phosphatebackbone

Cytidine

NH 2

H

O NH

N

Deamination of cytidine to uridine (spontaneous)

O

H

O NH

HN

sugar-phosphatebackbone

Uridine

NH 4+

H 2O

Cytidine

NH 2

H

O NH

N

sugar-phosphatebackbone

O

H

O NH

HN

sugar-phosphatebackbone

Uridine

NH 4+

H 2O

Recognized byDNA glycosylase

Uridine in DNA repaired by Base Excision Repair (BER)

MANY DNA GLYCOSYLASES EXIST

DIFFER IN SUBSTRATE SPECIFICITY

GENERALLY RECOGNIZE MONO-ADDUCT DAMAGE

Cytidine

NH 2

H

O NH

N

sugar-phosphatebackbone

O

H

O NH

HN

sugar-phosphatebackbone

Uridine

NH 4+

H 2O

Uracil-DNA glycosylase

sugar-phosphatebackbone

AP site

OH

H 2O

+

O

H

O NH

HN

H

Free uracil

Uridine in DNA repaired by Base Excision Repair (BER)

Thymine Dimer - a common DNA lesion

Nucleotide excision repair - Part I(bacteria)

Nucleotide excision repair - Part II(bacteria)

(uvrD)

Human nucleotide excision repair (NER)

•Xeroderma pigmentosum (XP) - an inherited disease in which patients show an extreme sensitivity to sunlight

•XP is a result of mutation of various genes involved in NER

Xeroderma Pigmentosum Society, Inc.Camp Sundown for XP children

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Mismatch repair

Primary source of DNA alterations arising during normal DNA metabolism is mispairing of bases during DNA synthesis

In eukaryotes, deamination of 5-methyl cytosine generates a thymine (and a T:G base pair) and is corrected by mismatch repair

A

A

Examples:

C

T

G

T

Question: Bases are not damaged, only incorrectly paired. How does the mismatch repair machinery determine which is the correct base and which is the incorrect base?

Determination of new strand (bacteria)

AT

CH3

CH3

DNA replication

New strand

AT

CH3

AT

CH3

New strand

+

CH3

CH3

Re-methylation (slow)

AT

CH3

AT

CH3+

CTAG

CH3

GATC

CH3

Mismatch repair (bacteria)

AT

CH3

CH3

G

T

DNA

replication CH3

Mismatch Recognition

mutS

G

T CH3

Binding ofmismatch factors

mutH

mutL

G

T CH3

G

T

Translocation ofmutL and mutH to

hemimethylated siteCH3

G

T CH3

Mismatch repair (bacteria)

AT

CH3

DNA synthesisby DNA Pol, SSB

& ligation

T

Exonucleasedigestion

CH3

Nicking of

non-methylated(new) strand

by mutH

G

T CH3

Nick

AT

CH3

CH3

Re-methylation

Hereditary nonpolyposis colon cancer (HNPCC)

•HNPCC is a hereditary cancer syndrome with individuals having increased incidence of colon cancer, ovarian cancer, and endometrial tumors

•Caused by defects in human mismatch repair genes that are homologous to bacterial mismatch repair genes

- Defects in hMSH2 (human mutS homolog) account for ~60% of HNPCC cases

- Defects in hMLH1 (human mutL homolog) account for ~30% of HNPCC cases

•Cells from HNPCC patients are 100-fold more mutable than normal patients

Reduction in error rate

Base pairing can lead to error frequency of ~10-1 -10-2 (i.e., errors per nucleotide incorporated)

DNA polymerase actions (polymerase specificity and 3’ --> 5’ proofreading) can lead to error frequency of ~10-5-10-6

Accessory proteins (e.g., SSBs) can lead to error frequency of ~10-7

Post replicative mismatch repair can lead to error frequency of ~10-10

Involvement of ATM and p53 in the cellular checkpoint response

M

G1

G2

G2/M(ATM, p53)

S phase(ATM, ATR, ...) G1/S

(ATM, p53)

S

Ionizingradiation

Severe damage

Apoptosis

AT is a genetic disorder with a incidence of 1 per 40,000 births

Ataxia Telangiectasia (AT)

Approx. 10% of individuals with AT develop neoplasms, such as Hodgkin’s disease, with most of these occurring with people less than 20 years of age

The overall cancer incidence in homozygotes is ~100-fold increased.

AT individuals have defects in gene encoding the checkpoint kinase ATM

The tumor suppressor p53The tumor suppressor p53

•The 'guardian of the genome’

•Functions as a sequence-specific transcription factor regulating a large number of genes

•The most frequently mutated gene in cancer

•Responsive to a wide array of signals that stress the cell including:

DNA damagehypoxiahyperproliferative signals emanating from oncogenes

p53-dependent apoptosis suppresses tumor growthp53-dependent apoptosis suppresses tumor growth

Choroid plexusepithelium

Van Dyke, 1994

p53 status is a determinant of tumor response to therapyp53 status is a determinant of tumor response to therapy

p53 +/+ p53 -/-

+ adriamycin)

Lowe, Science 266:807, 1994

tum

or v

olum

e (c

m3 )

+ adriamycin

Pathway of Carcinogenesis

Non-repairedmismatchDNA replication

Non-critical gene or non-coding

sequence

Cancer

Little or no effect on cell

viability

Essential region of

essential gene

Cell death through apoptosis

Potential for unregulated cell

growth

Additional mutations(genomic

instability)

Gene involved in growth

stimulation or tumor

suppression

Pathway of Carcinogenesis(colorectal cells)

Normalcell

Mutation of: PTGS2

(proto-oncogene)

p53

(tumorsuppressor)

(tumorsuppressor)

18q LOH

Carcinoma

Ras

(proto-oncogene)

LateAdenoma

APC

(tumorsuppressor)

EarlyAdenoma