Download Lecture 11

Most UV lesions are repaired by Nucleotide Excision Repair (NER)
Stalled replication forks may be bypassed by alternative (bypass) DNA polymerases
(REV1, REV3, RAD30)
Bypass polymerases have a cost
They are “error prone” on normal sequences
Replication
-Replication through ssDNA creates DSB
-DSB can arise spontaneously or by artificial means
-Ionizing radiation
-Mechanical force i.e. mitosis
-Incomplete action of topos
-endonucleases
-How does the cell deal with these DSB and what are possible outcomes???
Recombination as a source of genetic instability
A hallmark of cancer cells is their genetic instability
Loss of heterozygosity
Reciprocal and nonreciprocal translocations
Deletions
Truncations
Chromosome loss
Most of these types of instability may be explained by
various mechanisms of homologous and nonhomologous
recombination
In all probability the initiating lesion on DNA that leads
to these changes in chromosomes can be attributed to a
double-strand break (DSB)
Genome
instability
in tumor
cells
Truncations
Translocations
Inversions
Duplications
Amplifications
Abdel-Rahman et al. PNAS 98: 2538
When breaks in DNA cannot be repaired, bad things happen
Here, DT40 cells lack the
key recombination
protein, Rad51
Stalled replication forks may “break” (probably they are cut by an endonuclease)
Replication forks often stall at highly repetative sequences
Restriction fragment
size
shape
Stabilize Replication Forks
Replication fork “regression” Formation of a Holliday junction
Holliday junction formation
Extension of leading strand
Branch migration reverses
Holliday junction and allows
bypass of the UV-lesion
Holliday Junctions
• All base pairs made
• Cross Point (Branch can move)
• Resolved with or without help of factors
Holliday Junction
Replication fork “regression” Formation of a Holliday junction
RuvA, RuvB
Holliday junction formation
Extension of leading strand
Branch migration reverses
Holliday junction and allows
bypass of the UV-lesion
Helicase
Shape
B
RuvA
RuvB
GO TO:
http://www.sdsc.edu/journals/mbb/ruva.html
RuvC Necessary for resolution
• RuvA/B sufficient to resolve HJ with an end
nearby
• RuvC cleaves symmetrically on 2 homologous
DNA segments
The EMBO Journal (1997) 16, 1464–1472, doi: 10.1093/emboj/16.6.1464
Branch
Migration
Resolution of a Holliday Junction
Holliday Junction
Cleavage of a Holliday junction at a stalled
replication fork produces an intact template and a
broken-ended molecule
BIR
BIR
5’ to 3’ exonuclease
resection
Binding of Rad51
Strand invasion
Homology search
Re-establishment of a
replication fork
Branch migration
allows gap to be
filled in
Ends flipped over
for easy viewing
Another Holliday
junction
STRAND INVASION
Basic strand exchange
Replication Restart Can Lead to SCE
Recombination as a source of genetic instability
A hallmark of cancer cells is their genetic instability
Loss of heterozygosity
Reciprocal and nonreciprocal translocations
Deletions
Truncations
Chromosome loss
Most of these types of instability may be explained by
various mechanisms of homologous and nonhomologous
recombination
In all probability the initiating lesion on DNA that leads
to these changes in chromosomes can be attributed to a
double-strand break (DSB)
Genome
instability
in tumor
cells
Truncations
Translocations
Inversions
Duplications
Amplifications
Abdel-Rahman et al. PNAS 98: 2538
Eukaryotic recombination machinery
complex
• Large number of proteins
necessary
– Rad51, Rad52, 5 Rad51
paralogs (load Rad51)
– Rad54 – strand invasion
– BRCA1 and BRCA2
• Rad51, BRCA1/2 KOs
lethal
• Rad51 paralogs – less SCE
DSB
HOMOLOGOUS RECOMBINATION
Chromosomes and
Chromatids break other times
than during replication
Gene Conversion w/o Crossover
Gene Conversion w/ Crossover
Break-Induced Replication
Nonreciprocal translocation
Single-strand annealing
Gene amplification
NONHOMOLOGOUS RECOMBINATION
NH End-Joining
New telomere formation
Repaired BIR and Gene
Conversion
BIR – Non replicating cell
In humans. Telomerase is shut off at birth.
Telomeres get shorter and shorter every time DNA replicates.
People who have a deletion of one of the two telomerase RNA genes suffer from
dyskeratosis congenita (haplo-insufficiency)
Tumors arise by:
(a) re-activating telomerase
(b) ALT (alternative lengthening of telomeres)
ALT
• Extension telomeres by HR demonstrated
yeast
• Deleted telomerase enzyme
– Shorten 10nt/generation
– Die 30-50 generations
– Few telomerase independent survivors
• Eliminated Rad52
Gene Conversion - Synthesis dependent strand
annealing (SDSA)
Requires Rad51, Rad52, Rad54 etc.
Also requires PCNA and DNA
ploymerases
inducible
Cell knows which donor to choose – requires recombination enhancer
Restriction size differences at a and alpha site
Can look for presence of HJs
• Studied using 2D Gels hotspots
• Looking for differences novel spots not part of
replicating arc of DNA
• Cut out and use denaturing gel
– Each strand characterized
DSB
HOMOLOGOUS RECOMBINATION
Chromosomes and
Chromatids break other times
than during replication
Gene Conversion w/o Crossover
Gene Conversion w/ Crossover
Break-Induced Replication
Nonreciprocal translocation
Single-strand annealing
Gene amplification
NONHOMOLOGOUS RECOMBINATION
NH End-Joining
New telomere formation
Repaired BIR and Gene
Conversion
Hin gene
H1 gene Rh2 gene
OP H2 gene
H1 gene Rh2 gene
OP H2 gene
promoter
No transcription
P
promoter
Hin gene
P
Site-specific Recombination
Nicks on one pair of strands
Reciprocal strand exchange
(Holloiday junction)
Nicks on second pair of strands
Reciprocal strand exchange
Two examples: Hin and FLP
Site-specific recombination
One example: phase variation in Salmonella
Anti H1 antibody
eliminates 99.99%
Flagella composed of H1 flagellin
Survivors have alternative
H2 flagellin