Lecture 3, 4, ~5: DNA replication, repair and recombination Flashcards
How does gene conversion occur
DNA synthesis during homologous recombination
Repair of mismatches in regions of heteroduplex DNA
Methylated Cytosine
Problematic. Deamination results in T mismatched with G
Special DNA glycosylase repairs but is relatively ineffective
Only 3% of C’s are methylated, but they account for 1/3 of all point mutations associated with inherited disease
Helicase loading proteins
Cdc6
Cdt1
Two outcomes of holliday junction resolution
Crossing over (rare, only 2 events/chromosome) Gene conversion (90% of the time)
Reader-writer complex
Spreads parental patterns of histone modification
Homologous repair differences from non homologous
Uses daughter DNA duplex as template
No loss or alteration of DNA at repair site
Can repair other types of DNA damage
Gene conversion
Divergence from the expected distribution of alleles during meiosis
Resolution of holliday junction
Strands of helices are cleaved by endonuclease (RuvC)
Telomerase
Replicates chromosome ends in eukaryotes
Special sequence GGGTTA repeated x1000
Elongates the parental strand, then DNA polymerase copies onto new strand
Type II topoisomerase
Creates transient DS break of DNA.
Used at points of DNA where two double strand helices cross eachother
It can separate two interlocked DNA circles and prevent severe tangling problems that could arise during replication
Histone octamer breaks into what as replication fork arrives
H3-H4 tetramer- Randomly distributed to daughter duplexes
Two H2A-H2B dimers- Released from DNA
MutL
Mismatch repair protein
Scans for nick and triggers degradation of nicked strand
Sliding clamp
Keeps DNA polymerase on DNA when moving, releases with DS-DNA is encountered
Transcription coupled repair
RNA polymerase stops at lesion and directs repair machinery
Can work with BER, NER and other mechanisms
Brca1
Regulates processing of broken ends of chromosomes
Mutations lead to use of non-homologous end joining process
BER
Repairs single base pair changes
Involves DNA glycosylase, AP endonuclease, phosphodiesterase
How many base pairs in each turn of the DNA helix?
10bp
Cockaynes syndrome
Caused by defect in transcription coupled repair
Non homologous end joining differences from homologous
No-template required
Creates mutation at site of repair
Can also create translocations
Causes of DS DNA breaks
Ionizing radiation
Replication errors
Oxidizing agents
Depurination
N-glycosyl linkage is hydrolyzed, releasing a guanine or adenine- occurs spontaneously 5000x per day
Holliday junction strands cut in opposite directions
Crossing over
ORI (replication origin)
AT rich sequences attract initiator proteins
Must have binding site for ORC origin recognition complex
Must having binding site for proteins that help attract ORC
SS-DNA binding proteins
Bind single stranded DNA
Help stabilize unwound DNA
Prevent formation of hairpins
The DNA bases remain exposed
Brca2
Maintains Rad51 (RecA) Inactive until it is at site of damage Mutation causes RecA not to bind DNA to form invading strand
Holliday junction definition
DNA intermediate containing four DNA strands from two different helices
Checkpoints
DNA damage triggers pause of cell cycle
Blocks entry from G1–>S phase
Slows progression through S phase
Blocks transition from G2–>M phase
Clamp loader
Hydrolyzes ATP as it loads clamp. Stays near on the lagging strand to load clamp at each new fragment
AP endonuclease and phosphodiesterase
Cut phosphodiester backbone to remove sugar phosphate for repair in BER
NER
Can repair any bulky lesion
Multienzyme complex scans DNA for distortion in double helix instead of specific base change
Cleaves phosphodiester bond, DNA helicase peels lesion containing stand away
Non-homologous repair
Most common
Nuclease processes DNA ends
Ligase seals ends
Holliday junction strands cut the same way result in
Gene conversion
Replicative senescence
Daughter cells have defective chromosomes after many generations and stop dividing to guard against cancer
DNA glycosylase
BER enzyme
6 types, each recognize specific type of altered base and catalyzes its removal.
Cleaves glycosyl bond connecting base w/sugar
Mismatch repair
Removes almost all errors missed by proofreading by detecting distortion caused by mispairing
Human mutation rate
One nucleotide change per 10^8 nucleotides per generation
ATM protein
Kinase that generates intracellular signals to alert cell to DNA damage and upregulate DNA repair genes
In S phase, activated Cdks lead to:
Dissociation of helicase loading protein
Activation of helicase, unwinding of DNA
Loading of DNA polymerase, etc…
Histone chaperones
Reassemble histones after replication
Nucleases that generate 3’ invading strand are active in which phase(s)
S and G2-ensures a replicated chromosome or sister chromatid are most likely template for repair
Heteroduplex DNA
Double helix from DNA strands that originate from different molecules (strand from maternal homolog is base paired with strand from paternal homolog)
RecA
Binds the 3’ end of single stranded invading strand and directs it to a homologous sequence to form heteroduplex.
Requires at least 15 base pairs of homology
Homologous recombination occurs between, begins with what, has what intermediate steps
Paired maternal and paternal homologous chromosomes
Begins w/double stranded break
Strand invasion and double holliday junction formation follow
Translesion polymerase
Used to repair extensive damage, less accurate though
7 types, all lacking exonuclease proofreading ability
Adds only a couple nucleotides before real polymerase reassociates
Branch migration
Once strand invasion occurs, the point of exchange can move through branch invasion
Depurination repair shortcut
Begins directly with AP endonuclease, skips glycosylase
Mismatch repair uses what to distinguish correct strand?
In E. coli: It depends on methylation to distinguish new strand
In humans: Uses single strand breaks on lagging strand
DNA topoisomerase
Breaks phosphodiester bond to relieve supercoiling
T-loops
structures protect ends of chromosomes and distinguishes them from broken ones that need repair
Homologous recombination definition
Genetic exchange between a pair of homologous DNA sequences
Branch migration types
Spontaneous (can occur in both directions)
Catalyzed by special helicase (moves in one direction)
Exonucleolytic proofreading
Takes place immediately after incorrect base is added. Exonuclease clips off unpaired residue and DNA polymerase continues
DNA helicase
Unwinds DNA
Protein with 6 subunits.
Binds and hydrolyzes ATP, causing conformational change that propels it like a rotary engine, unwinding DNA
Type I topoisomerase
Creates single strand break of one phosphodiester bond, uses the other as a swivel point
Homologous repair
Nuclease processes 5’ ends
Damage repaired accurately using sister chromatid as template
Types of repair (4)
NER
BER
Transcription coupled repair
DS break repair (non homologous or homologous)
Mre11 Nuclease complex
Identifies DNA damage and processes ends of DS break in meiotic recombination
Homologous recombination uses
Repair of double-strand breaks
Exchange of genetic information to create new combinations of genetic sequences
Mechanical role in assuring accurate chromosome segregation
MutS
Mismatch repair protein.
Binds to mismatch
When are histone proteins synthesized
Mainly in S phase
Deamination
Cytosine changes to Uracil, occurs spontaneously 100x per day
ORC regulation
New ORC cannot be formed until next M phase resets cycle
