lecture 3 - homologous recombination

what is recombination

< variety or processes which involve the breakage and reunion of polynucleotides

< it increases genetic diversity

general or homologous recombination (HR)

< occurs between two homologous DNA duplexes - extensive sequence homology

< overall organisation of DNA doesn’t change

< within any part of the chromosome

< genes still in same position

specialised recombination

< occurs only between specific short sites of DNA

< requires short sequences of homology or doesn’t require homology

< often makes changes in the organisation of DNA (it changes place or transpons - jumping genes) or integration of a bacteriaphage into a bacterial chromosome which is a type of specialised recombination

homologous recombination in eukaryotes

< this typically occurs during meiosis I

< main role to increase genetic diversity

< during alignment, genetic material form the paternal and maternal copies of each chromosomes overlap causing a crossover and there is physical exchange of genetic material

what is homologous recombination essential for?

< evolution

< in prokaryotes: main role is dna repair system

< genomes are dynamic, restructure allows genetic diversity

homologous recombination steps 1 - 5

1) in first chromosome there is a DSB which can be spontaneous due to damage or induced in the cell due to meiosis, initiates recombination (blue is first chromosome, red is second chromosome)

2) when cell recognises DSB, exonuclease with 5’ to 3’ activity degradation of the 5’ ends, creates a 3’ overhang

3) one of the 3’ overhangs will invade into a homologous chromosome- called single strand invasion forming a D-loop structure as one DNA strand of second chromosome is displaced

4) heteroduplex is formed which dna strand from second chromosome - reunion of two strands is called recombinant joint which can move along the dna with a movement called branch migration - every time it moves, both chromosomes exchange more information between - increasing genetic diversity - hydrgoen bonds being broken and formed on the other side - breakage and reunion of base pairs - lots of mismatches in this process - homologous chromosomes are not identical so carry same genes but different alleles - so segments of dna are similar but not identical

5) Extension of the 3’ end by DNA polymerase using the second chromsome as a template creating more genetic diversity

homologous chromosomes steps 6 - 9

6) displaced D-loop pairs with the other chromosome and dna polymerase fills the gap

7) 5’ end of first chromosome will perform a second single strand invasion, creating a second recombinant joint

8) DNA ligase seals the nicks generating 4 complete DNA strands and 2 holiday junctions

9) resolution of the holiday junctions is a critical step in HR as dna strands are entangled and must be sepeated

< two possible outcomes depending on how holiday junctions are resolved: if both cut in the same axis this generates non crossover dna as not a big exchange of both chromosomes but if both cut in different axes this obtains a crossover recombinant dna molecules as half of the chromosome is from first chromosome so extends a big portion of dna so is a recombinant dna molecule

homologous recombination (steps III) - step 9

9) resolution of holiday junctions is final step in the process

< specialized enzymes (resolvases) cut the DNA to separate the interlinked chromosomes - this has two possible outcomes

1) non crossover products - if the two junctions are cut in the same orientation (axis), the original chromosome structure is restored, and there is no crossover of genetic material between the homologous chromosomes

2) crossover products - If the two junctions are cut in different orientations (axes), there is a crossover event, and the homologous chromosomes exchange segments of DNA

importance of recombination process

< helps ensure that broken DNA is repaired accurately using a homologous sequence as a template

< important during meiosis for generating genetic diversity as it allows for the exchange of genetic material between homologous chromosomes

gene conversion unfinished

< process by which one DNA sequence is replaced by a homologous sequence so they become identical e.g. from heterozygous (two chromosomes with two different alleles) exchange info so become homozygous as have same allele but exchange info in an unidirectional way - so one dna molecule exchanges info to another dna molecule -

< this happens after the resolution of the holiday junctions and after HR and after the mismatch repair system repairs on of the strands - if uses A dna strand as template, then won’t have gene conversion, but if uses A dna strand as template then both

< mismatch repair system fixes mutations of HR and depending how, maybe gene conversion happens

< nonreciprocal unidirectional transfer of genetic information from one donor to one acceptor DNA molecule

< happens in eukaryotes and prokaryotes

< important for evolution (e.g. selection of successful alleles) - so some alleles disappear with evolutions due to environmental changes so whole community survives so one allele appears more often for a bacterium, toxin needed to survive

< in exam, not part of HR process, so don’t include, it might or might not happen after HR

what is gene conversion?

< process by which one DNA sequence replaces a homologous sequence, such that the two sequences become identical

< e.g. in a heterozygous individual (two different alleles at the same locus), gene conversion can convert the heterozygous condition into a homozygous one (same allele on both chromosomes)

when does gene conversion occur?

< after HR, after the resolution of holiday junctions

< after HR, the heteroduplex DNA may contain mismatches (because the strands come from different sources, and they may not be perfectly complementary)

< the mismatch repair system acts on these mismatches to correct them

< gene conversion can occur during the DNA repair phase only when mismatches in the heteroduplex DNA are corrected by the mismatch repair system

< this means - one allele is converted to the sequence of the other allele, making two sequences identical

< may or may not happen after HR

the RecBCD pathway in E.coli

< HR process generating a 3’ single stranded overhang as a substrate for HR to perform single stranded invasion and all the steps in the previous slides

< then all the rest of the steps the same, just the beginning which is different in e.coli

what are Chi sites?

< hotspots which stimulate recombination

< sequences of 8 nucleotides (5’-GTCGGTGG-3’) every 5 to 10 kb

< recombination takes place around these chi sites

steps of the RecBCD pathway in E.coli unfinished

1) RecBCD complex binds to the DSB to fix the damage and then travels along the DNA

2) RecB (3’ to 5’ helicase) and RecD (5’ to 3’ helicase) unwinds the two DNA strands

3) at the same time RecB has exonuclease activity degrading one strand with 3’ to 5’ exonuclease activity (~1kb/s) until the Chi site - the complex stops at the Chi site as there is a 5’ overhang

4) RecB switches to endonuclease activity and cuts the other strand near the Chi site leaving a 3’ overhang

5) RecD detaches, ir produces a conformational change which abolishes RecB nuclease activity, only helicase

6) 3’ overhang is used as a substrate for homologous recombination to perform single stranded invasion, mediated by protein RecA

< RecA binds to and protects free 3’ overhang from degradation and mediates the formation of the heteroduplex during HR

RecA protein in bacteria unfinished

RecA essential for repair and maintenance of DNA.

Two types of activity (among others):

-- Activates the SOS response Stimulates protease activity

Promotes single strand invasion (base pairing between a singles strand DNA and its complement in a duplex molecule)

The strand invasion reaction requires three general conditions: one of the molecules has a

1) 3’ end,

2) single stranded region that it Is

3) complementary between molecules

what does RecA have?

< two DNA binding sites

1) primary site binds to ssDNA

2) secondary sites binds to dsDNA allowing heteroduplex formation

< RecA is a DNA dependant ATPase

< when RecA is bound to ATP it is active and has high affinity for DNA beginning single stranded DNA invasion

< hydrolysis of ATP to ADP decreases affinity to DNA and RecA is released from the heteroduplex DNA

< RecA is released from the heteroduplex DNA

RecFOR system in E. coli

< HR pathway which repairs SS gaps

< requires three proteins: RecF, RecO and RecR

< but if RecBCD is abolished, RecFOR can also repair DSBs and take over from the RecBCD system

< similar steps to RecBCD, it also requires RecA for strand invasion and DNA exchange

RuvABC proteins: Molecular rotor for branch migration watch recording

< branch migration and resolution of holiday junctions are catalysed by the RuvABC system

< RuvAB complex is a molecular rotor which rotates the DNA helices to allow branch migration (10-20 bp/second) using ATP as an energy source

< RuvA (tetramer