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 ?