Lecture 12 Flashcards
What does survival of a species depend on?
genetic diversity such that at least some population of the species can respond to unforeseen environmental pressures
Diversity is maintained by what 2 things?
maintained by both mutation, which alters a single gene, and by recombination, which redistributes the contents of the genome among various individuals during reproduction
What is recombination
any process by which DNA strands are broken and recombined to produce new combinations of alleles
What is recombination is important for?
an important mechanism for repair of double strand breaks and stalled replication forks, for generating a diverse immune repertoire, and for integration of viral genomes into host cell genomes
What does recombination involve
involves strand breakage and rejoining of DNA (crossing-over), usually for homologous duplexes (highly similar nucleotide sequences, homologous recombination).
What happens in homologous recombination
During meiosis, homologous chromosome pairs are transiently held together by recombination. The result is an exchange of genetic material between the pairs of sister chromatids. Recombination facilitates chromosomal segregation during cell division (because they help hold homologous chromosome pairs together) and increases the genetic diversity in the gametes.
4 examples of recombination?
- Repair of double strand DNA breaks
- Integration of viral genomes into host cell chromosomes (site-specific recombination)
- Repair of the replication forks stalled at the site of DNA damage
- Generating molecular diversity in antibodies
Describe the Holliday model of homologous recombination (a-g)
Proposed by Robin Holliday (1964)
homologous DNA strands align (gene A and a are homologous (2 alleles), genes B and b are homologous (+/- indicates antiparallel strand – 5’, 3’ ends)
strands of 2 DNA sequences are nicked by an endonuclease (c, d) nicked strands cross over to pair with complementary
strands on homologous duplex - “strand invasion”
nicks are sealed (ligated) - the crossover point is a 4-stranded structure called a Holliday junction
the Holliday junction can move in either direction of the duplex by unwinding of the original strand pair and rewinding of the new one - branch migration – can migrate in either direction – this results in a segment on one strand on each of the duplexes being swapped, but the ends (e.g., of the chromosome) remain intact – the Holliday junction connecting the two duplexes must be “resolved” – cleaved by a special endonuclease (a resolvase)
Holliday junction drawn differently to show how isomerization can occur
How does the Holliday junction resolve?
Resolves by strand breakage and rejoining
What product occurs during a horizontal cut?
horizontal cut: if the two strands that crossed over in step (b) break and rejoin the products will be non-
recombinant duplexes, each containing a heteroduplex region
(i.e., have the same AB or ab gene but
a segment of one strand is different from the original strand)
What product occurs during vertical cut?
if the strands that break are not those that originally invaded, the product will be two recombinant chromosomes (strands will have swapped homologous genes: Ab and aB ends), both having a recombinant heteroduplex region (i.e., a cross-over)
(l) cut strands are ligated by ligase
What forms when a template strand pairs with 2 different complementary DNA single strands (ssDNA)
A branch
How does the branch move
“Migrates” when base pairing to one strand (blue) is broken and replaced by base pairing to the other (red)
What does the RecA protein promotes
all the central steps in the homologous recombination process:
- pairing of two
- homologous DNAs
- strand invasion
- formation of Holliday intermediates
- branch migration
How does RecA mediates strand
exchange between single- stranded DNA (ssDNA) and double-stranded DNA (dsDNA)
- RecA protein polymerizes on ssDNA to form a nucleoprotein filament (1 RecA monomer for every 3 nucleotides)
- the RecA-coated ssDNA then invades the homologous duplex
What happens when the RecA/ssDNA nucleoprotein filament contacts a duplex DNA with a strand complementary (homology) to the bound ssDNA
RecA partially unwinds the duplex and, in an ATP-driven reaction, exchanges the ssDNA with the corresponding strand on the dsDNA
What happens as the RecA filament rotates about its axis
the duplex DNA is spooled in
two such strand exchange processes occur simultaneously in a Holliday junction, both mediated by ____
RecA in E. coli
eukaryotes contain proteins like ___ that are homologous to E. coli RecA and likely function in a similar manner
Rad51
Ds breaks occur from what?
- ionizing radiation (X-rays, gamma-rays, UV light), oxidative damage and other environmental factors
- a natural consequence of DNA replication at the site of a “lesion” (as in a DNA mismatch or damaged base that was not repaired by DNA repair mechanisms) – will result in collapse of the replication fork
Are ds breaks lethal or not?
are lethal to cells and must be repaired
Ds breaks are initiation sites for what? Thus results in?
initiation sites for homologous recombination – can result in diversity/adaptation, especially in bacteria, when recombination occurs with homologous segments of DNA taken up from other bacteria
ds breaks produce “blunt ends” – ____ processes these ends to produce ssDNA with a ___
- the RecBCD protein complex
- 3’-OH
___ is both a helicase and an exonuclease
RecBCD is both a helicase and an exonuclease
RecBCD binds what kind of DNA and where?
RecBCD binds linear DNA at a free (broken) end and moves inward along the duplex, unwinding the duplex with its helicase activity, and degrading both strands
Does RecBCD have exonuclease activities in what direction
has both 3’ → 5’ and 5’ → 3’ exonuclease activities
What happens when RecBCD encounters a Chi sequence
its 3’ → 5’ exonuclease activity slows and its 5’ → 3’ exonuclease activity increases, resulting in ssDNA with a 3’-OH end
- Rec A now binds to ssDNA
What does RecA mediate? Allows?
- Strand invasion of a homologous duplex,
- this process allows the break to be repaired using the homologous duplex
double-strand breaks (DSB) in a eukaryotic chromosome can be repaired by ?
homologous recombination using the sequence information of the homologous chromosome to reconstruct the original DNA (or by direct ligation using non-homologous or microhomology-mediated end joining)
Process of repairing DSB by homologous recombination?
- one of the strands at the break invades the homologous duplex DNA (see previous slide for E. coli mechanism) and uses it as a template to synthesize overlapping ends using DNA polymerase
- a “double Holliday intermediate” is formed and resolved by strand cleavage, strands are ligated
How many proteins are involved in eukaryotic DSB repair?
eukaryotic DSB repair involves many proteins
What is at almost every bacterial replication fork
an unrepaired lesion (e.g., a thymidine dimer, damaged base) or single-strand break (nick) is encountered
How often do damaged replication forks occr
damaged replication forks occur at least once per bacterial cell generation and 10X per eukaryotic cell division
What happens to DNA pol III when the replication fork is damaged
It cannot proceed past
Primary fxn of homologous recombination?
the primary function of homologous recombination may be to repair damaged replication forks
“stalled replication forks” are reactivated by ?
an elaborate coordination of all aspects of DNA metabolism – homologous recombination (HR)-mediated repair involves strand invasion, branch migration, Holliday junction formation, resolution
What are bacteriophage (phage)?
Viruses that infect bacteria
The (lambda) phage infects what?
infects E. coli and integrates its entire circular genome into a specific site on the E. coli chromosome using site- specific recombination
Difference btwn homologous recombination and site-specific homologous recombination?
Whereas homologous recombination can occur between any two homologous regions of a duplex pair, site-specific homologous recombination occurs at very specific sites in a DNA sequence, which are present in both the bacterial and the phage genomes.
site-specific homologous recombination are recognized by?
A recombinase enzyme
site-specific homologous recombination is driven by? Used for?
the highly specific DNA-recombinase interaction. Site-specific recombination is also used for programmed DNA rearrangements in embryonic development.
The lamda phage genome integrates via what recombination site?
via its attP recombination site into the homologous E. coli attB site with the aid of the lambda phage recombinase, integrase (INT)
What does this the lamda phage genome integrating result in?
Recombination results in two new hybrid attB/attP sites being formed, attL and attR, which the lambda phage integrase can also bind to catalyze excision of the lambda phage genome. Both reactions use integration host factor (IHF), which is encoded by the bacterium.
The Cre recombinase (another integrase) binds as __ to __?
) binds as a tetramer to both the attachment site on the phage genome and the integration site on the bacterial chromosome (which are homologous), induces strand invasion, Holliday junction formation, resolution of the Holliday junction, and strand ligation to recombine the two DNA chromosomes into one.
What is strand invasion
Nicked strands cross over to pair with complementary strands on homologous duplex
What is a Hollidary junction
The crossover point at the 4-stranded structure
What is branch migration
Holliday junction moving in either direction of the duplex by under winding the original strand and rewiring of the new one
- the branch migrates when base pairing to one strand is broken and replaced by base pairing to the other
What are non-recombinant duplexes
Each containing a heteroduplex region (have same AB or ab gene but one strand is diff from original)
What are recombinant chromosomes
Strands swapped homologous genes Ab and aB ends
What is a recombinant heteroduplex
Cross-over
Fxns of RecA protein?
- promotes the steps in homologous recombination (pairing of 2 homologous DNAs, strand invasion, formation of Holliday intermediates, branch migration)
- Mediates strand exchange between ssDNA and ddDNA
RedBC has both a ____ and an ____
- helicase and exonuclease (3’-> 5’ and 5’ -> 3’)
T or F? RecBCD binds linear DNA at free (broken end) and moves inward along duplex, unwinding duplex with its helical activity and degrading both strands
T
What happens when RecBCD encounters a chi sequence
its 3’->5’ exonuclease activity slows and its 5’-> 3’ exonuclease activity increases, resulting in ssDNA w/3’OH end
How does lambda phage infect E coli?
Integrates entire circular genome using site specific recombination
Which enzyme recognizes these site specific sites for homologous recombination
recombinase enzyme (integrase)
What does the recombination result in
2 new hybrid attB/attP sites being formed… attL and attR
Lytic phase genome vs lysogenic phage genome?
- Lytic phage genome doesn’t integrate into host cell chromosome. It is replicated extra-chromosaly. Assembled phage often kill host when released, killing cell
- Lysogenic phage genome integrate into host cell chromosome. Replicated along with host chromosome and passed onto daughter cells.
What do transposons encode
transposases (enzymes that catalyze transposition
Does transposition require DNA homology
No
Two classes of transposons?
Direct and replicative
What happens in direct transposition
Transposon segment moved from donor site to target site
What happens in replicative transposition
Transposon is copied and inserted at a separate site in the DNA
