DNA Recombination Mechanisms AHMP 5405

Objectives List the major classes of mobile genetic elements (we went over this before) Describe the process of general recombination Diagram the process of gene conversion via Holliday junctions Describe ways by which site-specific recombination can influence DNA rearrangement and genetic regulation

Recombination repair Present in prokaryotic and eukaryotic cells Only poorly understood We know it exists because UvrA- and RecA- cells are much more sensitive to UV than cells containing only one mutation

Why do chromosomes undergo recombination? Deleterious mutations would accumulate in each chromosome Recombination generates genetic diversity

Recombination ABCDEFGhijklmnoPQRSTUVWXYZ abcdefgHIJKLMNOpqrstuvwxyz ABCDEFGHIJKLMNOPQRSTUVWXYZ abcdefghijklmnopqrstuvwxyz

Mitotic and meiotic recombination Recombination can occur both during mitosis and meiosis Only meiotic recombination serves the important role of reassorting genes Mitotic recombination may be important for repair of mutations in one of a pair of sister chromatids

Recombination mechanisms Best studied in yeast, bacteria and phage Recombination is mediated by the breakage and joining of DNA strands

The Holliday model Two homologous duplexes are aligned Strand exchange leads to an intermediate with crossed strands This branch can move: Branch migration The branch is resolved by cleavage and sealing

Double strand break model

Double-strand breaks in DNA initiate recombination (part I)

Double-strand breaks in DNA initiate recombination (part II)

The cross-strand Holliday structure is an intermediate in recombination (part I)

The cross-strand Holliday structure is an intermediate in recombination (part II)

Initiation of recombination by the RecBCD enzyme

Branch migration and resolution of Holliday structures depends on Ruv proteins

Action of E. coli proteins in branch migration and resolution of Holliday structures Action of E. coli proteins in branch migration and resolution of Holliday structures

Chi structures When plasmids recombine figure eight structure is formed If the recombined plasmids are cut with a restriction enzyme a  (chi) is formed

Generation of a chi intermediate

Electron micrograph of the chi form

What does the Chi structure prove? The fact that each pair of arms is the same length shows that the circles are joined at homologous sites

Recombination between homologous DNA sites Recombination provides a means by which a genome can change to generate new combinations of genes Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is a mechanism for generating genetic diversity Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the sites

Cre protein and other recombinases catalyze site-specific recombination

The mechanism of Cre-loxP site- specific recombination

Site specific recombination Viruses and transposable elements often integrate their genomes into the host chromosome Site specific recombination is used by both eukaryotes and prokaryotes to regulate gene expression and to increase the organisms genetic repertoire

Site specific recombination

Mechanism of gene rearrangement

V(D)J recombination

Repair by End Joining (Recombination Repair)

DNA non-homologous end- joining (NHEJ) Predominant mechanism for DSB repair in mammals. Also exists in single-celled eukaryotes, e.g. Saccharomyces cerevisiae Particularly important in G0/G1

Homologous recombination Resection Rad50, Mre11, Xrs2 complex Strand invasion Rad52 Rad51; BRCA2 DNA synthesis    Ligation, branch migration, Holliday junction resolution DSB Non-homologous end-joining Ku70, Ku80 Ligation DSB DNA-PKcs Rad50, Mre11, Xrs2 complex “Cleaning up” of ends XRCC4/ Ligase IV

DNA-dependent protein kinase (DNA-PK) DNA-PK INACTIVE DNA-PK ACTIVE KINASE DNA

DNA-PK has three subunits INACTIVE ACTIVE DNA Ku70 Ku80 Ku70 Ku80 DNA-PKcs 69 kDa 83 kDa 470 kDa DNA-PKcs ATPADP X P Target sites: Ser/Thr-Gln

DNA-PK has three subunits INACTIVE ACTIVE DNA Ku70 Ku80 Ku70 Ku80 DNA-PKcs 69 kDa 83 kDa 470 kDa DNA-PKcs ATPADP X P … and is activated by DNA DSBs!

Multiple potential roles for Ku/DNA-PKcs in NHEJ

End-joining repair of nonhomologous DNA

NHEJ: links to cancer Status of NHEJ helps to define clinical radiosensitivity: Defects in DNA ligase IV associated with cells of a radiosensitive leukaemia patient (180-BR). Defects in DNA ligase IV associated with cells of a radiosensitive leukaemia patient (180-BR). Levels DNA-PK correlate with clinical outcome (cervical cancer). Levels DNA-PK correlate with clinical outcome (cervical cancer). Inherited or somatic defects in DNA-PK system may lead to cancer.

ATM: deficient in ataxia- telangiectasia (A-T) Human autosomal recessive disorder Progressive neurodegeneration Cancer predisposition Aspects of premature ageing Radiosensitivity Impairment in triggering cell cycle checkpoints in response to DNA DSBs

DNA-damage signalling is conserved from yeast to man S. pombe S. cerevisiaeH. sapiens Ddc1 Mec3 Rad17 P P Rad24 Rad9 Mec1 Tel1 Pds1 destruction Esp1 activation Cdc5 Crt1, Sml1? Chk1 Rad53 FHA P P Dun1 FHA P Rad9 Hus1 Rad1 P P Rad17 Brca1? ATR ATM Chk1 Chk2 FHA P P Rad9 Hus1 Rad1 P P Rad17 Crb2 Rad3 Tel1 Chk1 Cds1 FHA P P Cdc2 activation Cdc25 P Cdc2 (Cdk1) activation Cdc25C p53 P P G1-S Rad26 Lcd1 ?