Replication, Recombination and Repair Flashcards
What is recombination?
The shuffling of alleles between homologous chromosomes
What does recombination allow?
The selection of genes
A means of escape for favourable alleles and elimination for unfavourable alleles
Name 3 types of recombination
Homologous
Site specific
Transposition
What is homolgous recombination?
Responsible for meiotic crossing over and integration of transferred DNA into bacterial genomes
When does homologous replication occur in eukaryotes?
At the 4 strand stage of meiosis
What is the Holliday model?
A method of homologous recombination between double stranded DNA molecules with identical or nearly identical sequences.
What is the Holliday model applicable to?
Molecules with limited regions of homology or a single molecule with two separate regions of homology recombining with itself.
Describe the process of eukaryotic recombination.
Each sister chromatid contains duplex DNA
Homologous chromosomes pair and form bivalents
Pairing extends along chromosomes in the process of synapsis
Synaptonemal complex formed with homologous chromosomes side by side
Chromosomes separate but held together at chiasmata
Chromosomes condense, chiasmata remain, all 4 chromatids visible
Describe the Holliday model
Exchange of polynucleotides between 2 duplexes forms heteroduplex
Stabilised by base pairing between transferred strands
Gaps sealed by DNA ligase
What is resolution?
Spliced or patched
Cleaving across branchpoint reverts structure back into two individual double stranded DNA molecules
What is patched resolution?
A non-recombinant resolution due to cleavage in the same direction
What is spliced resolution?
A recombinant resolution due to cleavage in opposite directions
How does a hetroduplex region occur?
Two DNA molecules line up
Single stranded nicks appear at equivalent position in helix
What is the Meselson-Radding modification
Single-stranded nick in one helix which invades unbroken strand in the homologous position
Invasion produces D-loop
Cleavage of displaced strand generates heteroduplex
Where does recombination occur?
At double stranded breaks and single stranded gaps
Where do DSBs and SSGs come from?
DNA-damaging agents and normal DNA replication
What is the purpose of recombination?
Necessary to re initiate DNA replication
Repairs DSBs and rectifies aborted replication process
What does an exonuclease do in recombination?
Forms a double stranded break in the recipient strand
Digests 5’ side of nick forming 3’ overhang
What does RecA do?
Coats the 3’-ssDNA overhang
What are recombination specific helicases?
RecBCD and RecQ
What does RecBCD do?
Loads RecA protein onto overhang
What does RecQ do?
Works in conjunction with RecJ exonuclease to function at single stranded gaps
What happens when RecA fails to assemble?
RecF, RecO and RecR assemble
What is the presynaptic filament?
The nucleoprotein complex formed by the assembly of contigous RecA protein on ssDNA which undergoes homology search and strand invasion
What does the RuvAB complex do?
Branch migrates the crossover point
What does the RuvC protein do?
Holliday junction specific endonuclease which resolves the Holliday junction
What does the RuvABC complex do?
Recognises and cleaves the Holliday junction
What is oriC?
DNA replication in E.coli is origin dependent and must start from oriC
What is PriA?
A DNA replication protein that is required to reinitiate the replisome so Pol III can extend the nucleoprotein complex
What is gene conversion?
Distant markers from the crossover point are exchanged in a NON-reciprocal manner during recombination
One allele is converted to another with the exact base sequence represented at the converted site
What is initiation of replication?
The recognition of an origin and separation and stabilisation of parental strands
What is elongation?
Assembly of the replisome at the replication fork and synthesis of the daughter strands
What is termination?
Joining at the ends and separation of the chromosome
Describe the initiation process
Topoisomerase relaxes DNA
Initiator protein binds parental strand
DNA binds around initiator proteins, complex forms
Helicase untwists DNA causing local denaturation (needs ATP)
Primase binds helicase and denatured DNA
Primase activated by DNA helicase to synthesise short RNA primer
What is a primersome?
Helicase/primase complex
What does the primer do?
Serves as a substrate for action of DNA polymerase
Functions as pre-existing polynucleotide chain to which deoxynucleotides can be added
What does a DNA polymerase do?
Catalyses addition of deoxynucleotides to growing chain
What is a deoxynucleotide triphosphate?
Precursor to DNA polymerase which loses terminal pyrophosphate when nucleotide added
When is 3’ - 5’ proofreading exonuclease activity useful?
During bacterial replication
When is 5’ - 3’ exonuclease activity useful?
In nick translation in Pol I
How are okazaki fragments made?
DNA synthesis on lagging strand is discontinous
Primase synthesises RNA primer which is extended by DNA Pol III forming okazaki fragment
DNA Pol I uses nick translation to replace primer with DNA
DNA ligase seals the nick
How does proofreading work?
Incorrect nucleotide incorporated, polymerase stalls, free ss3’ end forms
3’ end enters exonuclease site and is cleaved to remove mismatch
How is the new strand recognised from the old in mismatch repair?
Recognises the methylation of selected A residues
Methylation of all A residues in the sequence GATC occurs in E.coli
Methylation doesn’t occur until some time after A is incorporated
GATC sequence behind replication fork not yet methylated therefore different from template strand
How is replication initiated in eukaryotes?
From multiple replicons which has its own origin so initiation can be regulated independently
Replicons close to each other initiate at the same time
What does Pol alpha do?
Initiates synthesis and s tightly associated with primase. Extends RNA primer by several nucleotides then dissociates
What does Pol delta do?
Primary DNA synthesising enzyme
What is PCNA?
Loads onto pol delta to make it processive so leading strand can synthesise
What is RFC?
The molecule that loads PCNA onto pol delta
How is the RNA primer removed in eukaryotes?
By nucleases FEN-1 and Rnase H1
What are direct repair systems?
They act directly on the damaged nucleotide to convert them back to the original form
How is alkylation damage reversed?
Direct transfer of alkyl group to enzyme polypeptide chain
What is photoreactivation?
In E.coli the enzyme DNA photolyase direectly repairs thymine dimers. When stimulated by light the enzyme binds the dimers and converts them to the monomeric nucleotide
What is base excision repair?
Removal of the damaged nucleotide followed by resynthesis to span the gap
Used to repair nucleotides with minor damage
DNA glycolyase cleaves base from sugar
Name 3 types of DNA glycolyases
Deaminated bases
Oxidation products
Methylated bases
What are apurinic and apyrimidinic sites?
Baseless sites created by DNA glycolyases. AP site is converted to a single nucleotide gap by an AP endonuclease
Sugar is cleaved
How is the gap filled?
In E.coli DNA pol I fills it
In mammals DNA pol beta fills it
What is short patch repair?
12 nucleotide segment is excised and patched
Initiated by a multienzyme complex called the UvrABC endonuclease
A trimer of 2 UvrA proteins and 1 UvrB binds the damaged site
UvrC binds UvrB (dimer)
UvrB cuts DNA downstream of damage site, UvrC cuts upstream of site
Intact section removed by DNA helicase II
What is nucleotide excision repair?
Cut and patch mechanism to remove lots of bulky lesions and chemical modifications
Will remove cyclobutly dimers by dark repair (no photoreactivation system)
What is transcription coupled repair?
RNA pol stalls at lesion
TFIIH released from transcription machinery
TFIIH binds at lesion site and DNA unwinds
Repair proteins fix lesion
What is mismatch repair?
mutS binds mismatch
mutS recruits mutL and mutH to bring GATC sequence close
mutH nicks unmethylated strand at GATC, methylated by Dam-methylase
New DNA hemimethylated, mismatch removed
Gap repaired by Pol III and ligase
What is the SOS response?
Induced to allow cell to survive lethal DNA damage
Increased capacity for DNA repair by inducing genes from repair pathways
What genes control the SOS response?
recA and lexA
What do checkpoints do?
Ensure only undamaged cells divide
What is RecG?
A branched DNA specific helicase that unwinds forked DNA to form four stranded Holliday junctions
What is DnaB?
A replicative helicase that unwinds the parental duplex ahead of the advancing replication machinery
What is DnaG?
A primase
What does PriA do in respect to RecG?
Reloads DnaB and DnaG onto the stalled replication fork DNA
How is a lesion repaired on a stalled replication fork?
Stalled forks unwound by helicase
Parental strands reanneal and daughter strands anneal
4 stranded Holliday junction formed
RuvAB and RuvC process junction
What are the 2 possible models for stalled fork lesion repair?
Chromosome breakage and recombination
Recombination without breakage
What is the model describing chromosome breakage and recombination?
Holliday junction processed bu RuvABC helicase-endonuclease complex
Junction cleaved by RuvABC
Lesion removed
D-loop formed by free duplex end combining with intact sister duplex by RecBCD and RecA
PriA binds free 3’ end
PriA loads DnaB/G on lagging strand to unwind
DnaG synthesises primers, replication continues
Why does chromosome breakage occur?
Although lethal, it is necessary to reinitiate a blocked replication fork
What is the model describing recombination without breakage?
Free duplex end acted on directly by RecA and RecBCD
Recombination with homologous sequence in parental duplex
Rec catalyses formation of D loop
How does RecG reinitiate a stalled replication fork?
RecG pulls parental strands across wedge with 2 channels
Channels can accommodate 2 ssDNA molecules
Leading and lagging strands displaced
Parental strands reanneal
Leading and lagging strands anneal to each other
Describe the process of PriA loading
PriA recognises branched DNA and catalyses loading of primosome onto lagging strand
At D loops lagging strand is ss and available to dnaB
PriA binds forks with leading strand at branch point
How do exonucleases aid the RecG chicken foot model?
Exonucleases digest the 5’ overhang of lagging strand
Removal of block by repair
Holliday junction winds back to replication fork
PriA loads productive replisome
What is Blooms syndrome?
A chromosomal disorder where there is an increases in spontaneous sister chromatid exchange leading to duplicated sequences and chromosome abberations
What is the importance of BLM helicase?
Resolves recombination intermediates to non-crossover recombinants
BLM and TOPO III alpha can process double Holliday junctions to non-crossover products
What is RecQ?
A family of helicases that can reinitiate stalled replication forks without forming a recombinant molecule
How do BLM and TOPO III alpha process dHJs?
BLM collapses 2 HJs into hemicatenane where the 2 single strands interlock
TOPO III alpha resolves the hemicatenanes
What happens when the BLM-TOPO III alpha pathway is not available?
Double Holliday junctions can only be resolved by becoming spliced or patched so there is an increased chance of recombinant molecules forming, creating fragmented chromosomes over time
What is RNAi?
An ancient evolutionary mechanism for genome defence in many organisms that functions to silence viruses and rogue genetic elements that make dsRNA intermediates
Also silences transposons
How does dsRNA silence genes?
By marking their mRNAs for destruction
What is Dicer?
An enzyme that catalyses the cleavage of dsRNA into siRNA
What is RISC?
RNA induced silencing complex which acts on siRNA and distinguishes between sense and antisense strands, degrading the sense strand
How is RISC activated?
By siRNAs unwinding by ATP
Describe the process of mRNA degradation by siRNAs
siRNAS incorporate mRNA, ATP and RISC molecules to form a nuclease/VIG/Ago-2/DFXR complex which can degrade mRNA
What do miRNAs do?
They silence genes at the protein synthesis stage by binding 3’UTR
How are Dicer and miRNAs linked?
pre-miRNA transported out of nucleus so Dicer can act on it
Dicer processes pre-miRNA hhairpins to mature ss form
Mature miRNA assembled into ribonucleoprotein complex
What is the full miRNA process?
Cellular gene produces Pri-miRNA
Drosha process Pri - Pre-miRNA
Dicer processes Pre - mature miRNA to form miRNP complex
miRNP complex binds 3’UTR through partially complementary region to inhibit transcription
What happens when the miRNA is exactly complementary?
The DNA is degraded
What are the differences between siRNA and miRNA?
siRNA target mRNA efficiently through degradation
siRNA silencing requires exact complementation with target mRNA
miRNA needs mRNA to have lots of partially complementary binding sites which work together
What does the splicing of introns affect?
Initial transcription Editing Polyadenylation of pre-mRNA Nuclear export Translational efficiency Decay of mRNA
How do introns enhance transcription?
Enhancers, repressors and nucleosome positioning elements within introns can influence efficiency
Spliceosomes assembling on newly transcribed intron can enhance transcription at initiation and elongation stages
How do introns affect RNA editing?
Required for modification of some exon sequences
A - I editing
What is an ADAR enzyme?
Adenine deaminase that acts on RNA
Converts adenosine to inosine in dsRNA
Describe the process of A - I editing
I is read as G during translation so A - I results in A - G
What is EJC?
The functional link between splicing and mRNA export
Describe the process of splicing and mRNA/nuclear export
Pre-mRNA is spliced
mRNA formed with EJC situated along strand
EJCs interact with Tap/p15 and nuclear pore complex components to facilitate mRNA export
Nuclear pore export occurs, EJC is remodelled
EJC situated on strand again
Translation occurs, EJC removed by ribosomes
