Double stranded break repair and homologous recombination

homologous chromosomes

similar

chromosome pair one from each parent

homologous recombination and homology directed repair involve

sequences that have high sequence identity (similarity)

DNA double stranded breaks often results from

replication of template containing a nick

chemicals can make nicks

ionizing radiation such as X-rays can directly break dsDNA

DS break repair types

-NHEJ Non-homologous end joining (rejoin ends w/o template)

-homology directed repair (use homologous DNA as template when sister chromatid is available)

-homologous recombination (mediates recombination crossing over of chromosomes in meiosis)

Non-homologous end joining (NHEJ)

only available pathway in G1 phase, error prone aka mutagenic

occurs by simple ligation of ends

or

remove a few nucleotides to make overhangs to pair, loose some info

Homology-directed repair

begins with DNA resection to generate 3’ overhangs which find homology

creates d-loop in image 3

homologous recombination can result in

crossover or non crossover

starts with D-loop

DNA cut at junctions

double Holliday junction formed and cleaved

homologous recombination involves?

formation and cleavage of Holliday Junctions

can cut two ways

When homology directed repair uses homologous chromosome as a template what can happen?

gene conversion

when sister chromosomes are not available

heteroduplexes

formed during recombination

can lead to gene conversion

have some bp mismatches

repaired o replicated to generate mother or father sequence

In yeast regulated gene conversion mediates

mating type determination

RecA and Rad1

RecA (bacteria) and Rad51 (eukaryotes) are critical for homologous-directed repair and homologous recombination

loaded on single stranded DNA to help pairing and forming D-loop

in vivo also need BRCA1 and BRCA2

Strand exchange assay

same sequence single strand circular and linear double strand put together with Rec A and over time nicked circle accumulates

shows strand displacement activity in RecA

mitotic recombination leads to

genetic exchange between similar DNA sequences

usually in meiosis

mitotic recombination between repeated sequences leads to

genome rearrangements

intrachromosomal recombination → deletions

repeated sequences → duplications and deletion

repeats on diff chromosomes can lead to translocations

Loss of heterozygosity (LOH) of tumor suppressor genes allows

growth of cancer, major driver of cancer

loosing checkpoint genes and DNA repair genes

LOH can result from

repair of ds break through gene conversion or Break Induced Replication (BIR)

or mitotic recombination in diploids

homologous recombination can be used to

knock out or alter specific genes