DNA replication and repair - Lecture 3
Jim Borowiec
September 28, 2006
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Overview of DNA replication
Telomere
Centromere
Telomere
DNA chromosome
Origin of
DNA replication
Specialized elements termed
'origins of DNA replication’
occur many times on a
chromosome
Initiation of DNA replication
from origin of replication
generates structures termed
'DNA replication bubbles'
End replication problem
3’
5’
DNA replication
(from internal regions of the chromosome)
by leading strand synthesis
3’
5’
3’
+
5’
by lagging strand synthesis
RNA primer
iDNA
Processing
loss of DNA
3’
DNA
5’
replication
3’
5’
After multiple rounds of DNA replication, genetic information will be lost
Replication of telomeres
•Telomeres contain many copies of a specific DNA repeat
•Involves special RNA-containing polymerase called telomerase
(TTAGGG)nTTAGGGTTAGGG-3’
(AATCCC)nAATCCC-5’
CCAAUCCC
Telomerase
5’
RNA template
(TTAGGG)nTTAGGGTTAGGGTTAGGG-3’
(AATCCC)nAATCCC
CCAAUCCC
5’
•Telomerase adds one or more copies of the TTAGGG repeat, preventing DNA loss
Telomerase needed for cell immortalization
Most somatic cells do not have telomerase activity
Telomere length
Germ line cells
Somatic cells
‘Hayflick limit’
p53 mutation
Telomerase activation
Widespread cell death
Senescence
Telomere stabilization
Crisis
Cell Divisions
Mechanisms to repair damaged DNA
or 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
A. Base deamination
(ex: cytosine is converted to uracil at a rate of ~100 bases per human cell per day)
B. Loss of bases - depurination and depyrimidation
(~5000 purines are lost per human cell per day)
C. Oxidative damage to bases
(life span of an organism is inversely correlated with
metabolic rate/DNA oxidation)
2. Environmental damage
A. Radiation (ionizing and ultraviolet)
B. Chemical agents (e.g., benzo[a]pyrene)
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 recruit
additional repair
factors
Deamination of cytidine to uridine (spontaneous)
NH 2
H
N
O
O
NH 4 +
H
HN
H
N
H 2O
O
H
N
sugar-phosphate
backbone
sugar-phosphate
backbone
Cytidine
Uridine
Uridine in DNA repaired by
Base Excision Repair (BER)
NH 2
H
N
O
O
NH 4 +
H
HN
H
N
H 2O
O
H
N
sugar-phosphate
backbone
sugar-phosphate
backbone
Cytidine
Uridine
Recognized by
DNA glycosylase
MANY DNA GLYCOSYLASES EXIST
DIFFER IN SUBSTRATE SPECIFICITY
GENERALLY RECOGNIZE MONO-ADDUCT DAMAGE
Uridine in DNA repaired by
Base Excision Repair (BER)
NH 2
H
N
O
O
NH 4 +
H
HN
H
N
H
O
H 2O
sugar-phosphate
backbone
N
sugar-phosphate
backbone
Cytidine
Uridine
H 2O
Uracil-DNA glycosylase
OH
sugar-phosphate
backbone
AP site
O
+
H
HN
O
H
N
H
Free uracil
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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The program schedule is 9:00 p.m. to 5:00 a.m. to maximize night time
hours for play and minimize need for protective arrangements.
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
Examples:
A
C
G
A
T
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)
A
T
CH3
CH3
GATC
CTAG
CH3
CH3
DNA replication
New strand
A
T
CH3
+
A
T
CH3
New strand
Re-methylation (slow)
A
T
CH3
CH3
+
A
T
CH3
CH3
Mismatch repair (bacteria)
A
T
CH3
DNA
G
CH3
replication
T
CH3
Mismatch
Recognition
mutS
G
T
mutH
Binding of
mismatch factors
mutL
G
T
CH3
CH3
Translocation of
mutL and mutH to
G
hemimethylated site
T
CH3
Mismatch repair (bacteria)
G
T
CH3
Nick
Nicking of
G
non-methylated
(new) strand
by mutH
T
CH3
Exonuclease
digestion
T
CH3
DNA synthesis
by DNA Pol, SSB
& ligation
A
T
CH3
CH3
Re-methylation
A
T
CH3
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
G2/M
(ATM, p53)
Ionizing
radiation
M
G2
G1
S
S phaseSevere damage
(ATM, ATR, ...)
Apoptosis
G1/S
(ATM, p53)
Ataxia Telangiectasia (AT)
AT is a genetic disorder with a incidence of 1 per 40,000 births
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 p53
•The 'guardian of the genome’
•The most frequently mutated gene in cancer
•Functions as a sequence-specific transcription factor
regulating a large number of genes
•Responsive to a wide array of signals that stress the cell
including:
DNA damage
hypoxia
hyperproliferative signals emanating from oncogenes
p53-dependent apoptosis suppresses tumor growth
Choroid plexus
epithelium
Van Dyke, 1994
p53 status is a determinant of tumor response to therapy
+ adriamycin
p53 -/-
+ adriamycin
)
tumor volume (cm3)
p53 +/+
Lowe, Science 266:807, 1994
Pathway of Carcinogenesis
DNA replication
Non-repaired
mismatch
Cancer
Non-critical gene or
non-coding sequence
Little or no effect
on cell viability
Essential region of
essential gene
Cell death through
apoptosis
Gene involved in
growth stimulation
or tumor
suppression
Potential for
unregulated cell
growth
Additional mutations
(genomic instability)
Pathway of Carcinogenesis
(colorectal cells)
(tumor
suppressor)
(protooncogene)
APC
PTGS2
Mutation of:
Normal
cell
Early
Adenoma
(protooncogene)
Ras
(tumor
suppressor)
(tumor
suppressor)
p53
18q LOH
Late
Adenoma
Carcinoma