DNA Recombination DNA Recombination MechanismsMechanisms

AHMP 5405AHMP 5405

ObjectivesObjectives

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

Recombination repairRecombination repairPresent in prokaryotic and eukaryotic cellsPresent in prokaryotic and eukaryotic cells

Only poorly understoodOnly poorly understood

We know it exists because UvrA- and RecA- We know it exists because UvrA- and RecA- cells are much more sensitive to UV than cells are much more sensitive to UV than cells containing only one mutationcells containing only one mutation

Why do chromosomes undergo Why do chromosomes undergo recombination?recombination?

Deleterious mutations would accumulate in Deleterious mutations would accumulate in each chromosomeeach chromosome

Recombination generates genetic diversityRecombination generates genetic diversity

RecombinationRecombination

ABCDEFGhijklmnoPQRSTUVWXYZabcdefgHIJKLMNOpqrstuvwxyz

ABCDEFGHIJKLMNOPQRSTUVWXYZabcdefghijklmnopqrstuvwxyz

Mitotic and meiotic recombinationMitotic and meiotic recombination

Recombination can occur both during Recombination can occur both during mitosis and meiosismitosis and meiosis

Only meiotic recombination serves the Only meiotic recombination serves the important role of important role of reassorting genesreassorting genes

Mitotic recombination may be important for Mitotic recombination may be important for repair of mutationsrepair of mutations in one of a pair of sister in one of a pair of sister chromatidschromatids

Recombination mechanismsRecombination mechanisms

Best studied in yeast, bacteria and phageBest studied in yeast, bacteria and phage

Recombination is mediated by the Recombination is mediated by the breakage and joining of DNA strandsbreakage and joining of DNA strands

The Holliday model The Holliday model

Two homologous duplexes are alignedTwo homologous duplexes are aligned

Strand exchange leads to an intermediate with Strand exchange leads to an intermediate with crossed strandscrossed strands

This branch can move: Branch migrationThis branch can move: Branch migration

The branch is resolved by cleavage and sealingThe branch is resolved by cleavage and sealing

Double Double strand strand break break modelmodel

Double Double strand strand break break modelmodel

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

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

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

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

Initiation of recombination by the Initiation of recombination by the RecBCD enzymeRecBCD enzyme

Branch migration and resolution of Branch migration and resolution of Holliday structures depends on Ruv Holliday structures depends on Ruv

proteinsproteins

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

structuresstructures

Chi structuresChi structures

When plasmids recombine figure eight When plasmids recombine figure eight structure is formedstructure is formed

If the recombined plasmids are cut with a If the recombined plasmids are cut with a restriction enzyme a restriction enzyme a (chi) is formed (chi) is formed

Generation of a Generation of a chi intermediatechi intermediate

Electron Electron micrograph of the micrograph of the

chi formchi form

What does the Chi What does the Chi structure prove?structure prove?

The fact that The fact that each pair of each pair of arms is the arms is the same length same length shows that the shows that the circles are circles are joined at joined at homologous homologous sitessites

Recombination between homologous Recombination between homologous DNA sitesDNA sites

Recombination provides a means by which a genome can Recombination provides a means by which a genome can change to generate new combinations of geneschange to generate new combinations of genes

Homologous recombination allows for the exchange of blocks Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is of genes between homologous chromosomes and thereby is a mechanism for generating genetic diversitya mechanism for generating genetic diversity

Recombination occurs randomly between two homologous Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sequences and the frequency of recombination between two sites is proportional to the distance between the sitessites is proportional to the distance between the sites

Cre protein and other recombinases Cre protein and other recombinases catalyze site-specific recombinationcatalyze site-specific recombination

The mechanism of Cre-The mechanism of Cre-loxPloxP site- site-specific recombinationspecific recombination

Site specific recombinationSite specific recombination

Viruses and transposable elements often Viruses and transposable elements often integrate their genomes into the host integrate their genomes into the host chromosomechromosome

Site specific recombination is used by both Site specific recombination is used by both eukaryotes and prokaryotes to regulate eukaryotes and prokaryotes to regulate gene expression and to increase the gene expression and to increase the organisms genetic repertoireorganisms genetic repertoire

Site specific Site specific recombinationrecombination

Mechanism Mechanism of gene of gene

rearrangementrearrangement

V(D)J recombinationV(D)J recombination

V-DJ Recombination

Transcription, Splicing

V-J Recombination

Ig/ T-cell Receptor mRNA

5'3'

"JOINING" SEGMENTS

"CONSTANT" REGION

"DIVERSITY" SEGMENTS

"VARIABLE" SEGMENTS

Repair by End Joining Repair by End Joining (Recombination Repair)(Recombination Repair)

DNA non-homologous end-DNA non-homologous end-joining (NHEJ)joining (NHEJ)

Predominant mechanism for DSB repair in Predominant mechanism for DSB repair in mammals.mammals.

Also exists in single-celled eukaryotes, e.g. Also exists in single-celled eukaryotes, e.g. Saccharomyces cerevisiaeSaccharomyces cerevisiae

Particularly important in G0/G1Particularly important in G0/G1

Homologous recombination

ResectionRad50, 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 DNA-dependent protein kinase (DNA-PK)kinase (DNA-PK)

DNA-PK

INACTIVE

DNA-PK

ACTIVE KINASE

DNA

DNA-PK has three subunitsDNA-PK has three subunits

INACTIVE ACTIVE

DNA

Ku70Ku80

Ku70Ku80

DNA-PKcs69 kDa

83 kDa

470 kDa

DNA-PKcs

ATP ADP

X

P

Target sites: Ser/Thr-Gln

DNA-PK has three subunitsDNA-PK has three subunits

INACTIVE ACTIVE

DNA

Ku70Ku80

Ku70Ku80

DNA-PKcs69 kDa

83 kDa

470 kDa

DNA-PKcs

ATP ADP

X

P

… and is activated by DNA DSBs!

Multiple potential roles for Multiple potential roles for Ku/DNA-PKcs in NHEJKu/DNA-PKcs in NHEJ

End-joining repair of nonhomologous End-joining repair of nonhomologous DNADNA

NHEJ: links to cancerNHEJ: links to cancer

Status of NHEJ helps to define clinical Status of NHEJ helps to define clinical radiosensitivity:radiosensitivity: Defects in DNA ligase IV associated with cells Defects in DNA ligase IV associated with cells

of a radiosensitive leukaemia patient (180-of a radiosensitive leukaemia patient (180-BR).BR).

Levels DNA-PK correlate with clinical outcome Levels DNA-PK correlate with clinical outcome (cervical cancer).(cervical cancer).

Inherited or somatic defects in DNA-PK Inherited or somatic defects in DNA-PK system may lead to cancer.system may lead to cancer.

ATM: deficient in ataxia-ATM: deficient in ataxia-telangiectasia (A-T)telangiectasia (A-T)

Human autosomal recessive disorderHuman autosomal recessive disorder

Progressive neurodegenerationProgressive neurodegeneration

Cancer predispositionCancer predisposition

Aspects of premature ageingAspects of premature ageing

RadiosensitivityRadiosensitivity

Impairment in triggering cell cycle Impairment in triggering cell cycle checkpoints in response to DNA DSBscheckpoints in response to DNA DSBs

DNA-damage signalling is DNA-damage signalling is conserved from yeast to manconserved from yeast to man

S. pombe S. cerevisiae H. sapiens

Ddc1

Mec3

Rad17P P

Rad24

Rad9

Mec1 Tel1

Pds1 destructionEsp1 activation

Cdc5Crt1, Sml1?

Chk1 Rad53FHA FHA

PP

Dun1FHAP

Rad9

Hus1

Rad1P P

Rad17

Brca1?

ATR ATM

Chk1 Chk2FHA

PP

Rad9

Hus1

Rad1P P

Rad17

Crb2

Rad3 Tel1

Chk1 Cds1FHA

PP

Cdc2 activation

Cdc25

P

Cdc2 (Cdk1) activation

Cdc25C

p53P

P

G1-S

Rad26 Lcd1 ?