Lecture 5 DNA Recombi 1 Exchange Flashcards
DNA damage: spontaneous
Spontaneous:
Spontaneous degradation of DNA (loss of bases or amino groups from bases)
Metabolic products mispairing ( e.g. reactive oxygen species such as 8-oxoguanine can mispair w/adenine)
misincorporation of bases or errors by DNA polymerases (e.g. mismatches) and DNA repair
DNA damage: induced
Misrepair of damage caused by radiation ( UV light, X rays, gamma rays, alpha and beta particles from nuclear decay)
Methylation of bases
Crosslinking reagents (e.g. cis-platin DNA crosslinking)
Intercalated molecules
Different types of DNA damage cause different types of lesion
These are repaired in different ways
Outcome:
1)cell survival and resumption of normal cell life cycle
2) cell death
3) malignant transformation (in human cells)
Survival response is complex with many signalling pathways
Homologous or genetic recombination
Conserved genetic identity:
-aligns chromosomes at meiosis
- repairs DNA breaks
- restores stalled replication forks
Generates genetic diversity:
-rearranges genes within genome
-exchanges homologous partners
-incorporates foreign DNA
- contributes to acquisition of new traits
Glossary
Recombination
- exchange of genetic info
Crossover/splice/recombinant
- recombi where flanking markers are exchanged e.g. chromosome w/g allele now joined to a partner with diff b allele
Non-crossover/patch/recombinant
Recombination where flanking markers are not exchanged though heteroduplexes still form
Homoduplex
DNA molecule composed of 2 strands each from same parent chromosome
Heteroduplex
DNA molecule composed of 2 strands each from a different parent chromosome
Gene conversion
A mismatched DNA sequence from one heteroduplex DNA strand is replaced with a sequence complementary to the other strand resulting in aberrant gamete ratios in meiosis (e.g. 3:1) depending on strand used as template for repair, wild-type or mutant alleles will be inherited
DNA recombination
Homologous or genetic recombination - repair system that helps recover from breaks that arise in chromosomal DNA. Crucial for dsDNA break repairs. Partner chromosomes found and ss exchanged to form DNA branched structures. Exchanges often provide a 4’ end to prime replication restart or offer template to copy any genetic info that might be lost
Models of homologous recombination: Holliday
1964 Robin Holliday proposed model to explain recombinants observed in Ascomycete fungi, Sordaria fimicola and Neurospora Crassa
Initiate with nick at exact same place in each DNA duplex. Suggesting regions of heteroduplex DNA would be symmetrical i.e. same amount of heteroduplex DNA at same plac on both parental chromosomes. Experiments w/yeast disproved this.
Models of homologous recombination: Meselson and Radding
1975 proposed a nick in one chromosome primes DNA synthesis from 3’ end that displaces a strand to form an invasive ssDNA tail that ultimately yields a Holliday junction (asymmetry in heteroduplex occurs)
Models of homologous recombination: Szostak et al.
1983 proposed a model where recombi initiated at dsDNA break (DSB) and formation of 2 Holliday junctions
Repairing dsDNA break by homologous recombination
In early models initiation events assumed to be at SS nicks but it was discovered that yeast transformation is stimulated 1000 fold when a dsDNA break is introduced into circular donor plasmid. Ds break repair model proposed by J. Szostak, T. Orr-Weaver, Rodney Rothstein and Frank Stahl
DS break repair (DSBR) and synthesis dependent strand annealing (SDSA)
DSB’s can be repaired by several homologous recombination (HR) mediated pathways including DSBR and SDSA
Repair initiated by resection ( degradation of one strand) of a DSB to provide 3’ ssDNA overhangs
Strand invasion by the overhangs into homologous duplex sequence (partner undamaged chromosome) is followed by DNA synthesis at invading end.
After strand invasion and synthesis the second DSB can be captured to form intermediate with 2 Holliday junctions
After gap repair DNA synthesis and ligation structure is resolved at HJ’s as cross/non cross over
Alternatively reaction can proceed to SDSA by strand displacement, annealing of extended ss end to ssDNA on other break end followed by gap-filling DNA synth and ligation. Repair product from SDSA always non crossover
Synaptic stages of recombination
Pre-synapsis: events take place that are needed for initiating recombination including formation of gaps,ss/ds breaks and exposure of ss regions
Synapsis: homologous strands are paired up and strand exchange takes place to produce 3- and then 4- stranded branch chain intermediates
Post-synapsis: 4 stranded Holliday junction is cut to give crossover or non-crossover products. Branch migration: moves the branch along the DNA.
Resolution: strand cleavage at HJ to separate linked chromosomes
Mismatch repair: corrects any base pair mismatches
In bacteria initial steps of recombi involve RecBCD and RecA proteins
Bacterial RecBCD and end resection (pre-synapsis)
Rec B and RecD are helicases that travel along each of the strands at the DNA break. Rec B moves 3’5’ direction and Rec D 5’3’ on opposite strand - thus they unwind DNA as a bipolar helicase. RecB has a nuclease domain that cuts both DNA strands initially as it passes through the Rec BCD complex.
Upon encountering a chi site (chi=crossover hotspot instigator - sequence of 8 nucleotides - GCTGGTGG-3’) cutting of the 3’ strand ceases but accelerates on 5’ strand. Rec B directs loading of RecA onto 3’ end and degrades only 5’ strand
Bacterial RecA homologous pairing and strand exchange ( synapsis)
RecA central to bacterial recombi process it’s a 37842 Da (352 amino acid) protein that binds ssDNA. RecA polymerises on exposed ssDNA to form helical nucleoprotein filament that can be seen by electron microscopy.
RecA nucleoprotein filament catalyzes homologous DNA pairing and strand exchange stages of recombi
RecA polymerises on ssDNA overhang generated by RecBCD and initiates a search for a homologous duplex. Strand exchange generates 3 stranded displacement (D) loop. Which can be extended to form 4 strand Holliday junction.
The right handed helical nucleoprotein filament promotes homologous pairing to form synaptic complex (dix recA subunits per helical turn) ssDNA within the filament is underwound and extended to 1.5 times B-form DNA which promotes homologous pairing and strand exchange reactions (strand exchange involves triple helix)
Key points
dsDNA breaks arise by exposure to ionizing radiation
Ends of break are processed by RecBCD exonuclease ultimately generating 3’ ssDNA overhangs
RecA is loaded on exposed ssDNA and initiates search for undamaged homologous chromosome
RecA mediates homologous pairing and strand exchange to form a D-loop which can be extended to generate a HJ
If this reaction occurs at both ends of the break then strand exchange results in 2 HJ which are resolved to gen cross/non crossover products
