Transcript DNA Recombination Mechanisms

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 RecAcells 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
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
Chi structures
When plasmids recombine figure eight
structure is formed
If the recombined plasmids are cut with a
restriction enzyme a c (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 sitespecific 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
5'
"VARIABLE"
SEGMENTS
"DIVERSITY"
SEGMENTS
V-J Recombination
V-DJ Recombination
Transcription, Splicing
Ig/ T-cell Receptor mRNA
"JOINING"
SEGMENTS
"CONSTANT"
REGION
3'
Repair by End Joining
(Recombination Repair)
DNA non-homologous endjoining (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
Non-homologous end-joining
DSB
Rad50, Mre11,
Xrs2 complex
DSB
Resection
Rad52
Ku70, Ku80
DNA-PKcs
Strand invasion
Rad50, Mre11,
Xrs2 complex
Rad51; BRCA2
“Cleaning up”
of ends
DNA synthesis




Ligation, branch migration,
Holliday junction resolution
XRCC4/
Ligase IV
Ligation
DNA-dependent protein
kinase (DNA-PK)
DNA
DNA-PK
DNA-PK
INACTIVE
ACTIVE
KINASE
DNA-PK has three subunits
DNA
Ku70
Ku70
X
Ku80
P
DNA-PKcs
Ku80
69 kDa
83 kDa
DNA-PKcs
ATP
ADP
470 kDa
INACTIVE
Target sites: Ser/Thr-Gln
ACTIVE
DNA-PK has three subunits
DNA
Ku70
Ku70
X
Ku80
P
DNA-PKcs
Ku80
69 kDa
83 kDa
DNA-PKcs
ATP
ADP
470 kDa
INACTIVE
ACTIVE
… 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).
Levels DNA-PK correlate with clinical
outcome (cervical cancer).
Inherited or somatic defects in DNA-PK
system may lead to cancer.
ATM: deficient in ataxiatelangiectasia (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. cerevisiae
S. pombe
P
Hus1
Rad9
P
Rad17
Mec3
Ddc1
Crb2
Rad1
P
Rad26
Rad3
Tel1
P
H. sapiens
Rad24
Hus1
Rad9
Rad9
Rad17
P
Lcd1
Mec1
Tel1
P
Rad17
Brca1?
Rad1
P
?
ATR
ATM
P
P
Chk1
P
FHA
Cds1
P
Chk1
P
FHA
Rad53 FHA
P
P
Chk1
FHA
P
p53
Chk2
P
FHA
Dun1
Cdc25
Cdc2 activation
Cdc25C
Pds1 destruction
Esp1 activation
Cdc5
Crt1, Sml1?
Cdc2 (Cdk1)
activation
G1-S