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Genes and Genomes > Replication, Recombination and Repair > Flashcards

Replication, Recombination and Repair Flashcards

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1
Q

What is recombination?

A

The shuffling of alleles between homologous chromosomes

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2
Q

What does recombination allow?

A

The selection of genes

A means of escape for favourable alleles and elimination for unfavourable alleles

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3
Q

Name 3 types of recombination

A

Homologous
Site specific
Transposition

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4
Q

What is homolgous recombination?

A

Responsible for meiotic crossing over and integration of transferred DNA into bacterial genomes

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5
Q

When does homologous replication occur in eukaryotes?

A

At the 4 strand stage of meiosis

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6
Q

What is the Holliday model?

A

A method of homologous recombination between double stranded DNA molecules with identical or nearly identical sequences.

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7
Q

What is the Holliday model applicable to?

A

Molecules with limited regions of homology or a single molecule with two separate regions of homology recombining with itself.

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8
Q

Describe the process of eukaryotic recombination.

A

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

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9
Q

Describe the Holliday model

A

Exchange of polynucleotides between 2 duplexes forms heteroduplex
Stabilised by base pairing between transferred strands
Gaps sealed by DNA ligase

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10
Q

What is resolution?

A

Spliced or patched

Cleaving across branchpoint reverts structure back into two individual double stranded DNA molecules

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11
Q

What is patched resolution?

A

A non-recombinant resolution due to cleavage in the same direction

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12
Q

What is spliced resolution?

A

A recombinant resolution due to cleavage in opposite directions

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13
Q

How does a hetroduplex region occur?

A

Two DNA molecules line up

Single stranded nicks appear at equivalent position in helix

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14
Q

What is the Meselson-Radding modification

A

Single-stranded nick in one helix which invades unbroken strand in the homologous position
Invasion produces D-loop
Cleavage of displaced strand generates heteroduplex

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15
Q

Where does recombination occur?

A

At double stranded breaks and single stranded gaps

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16
Q

Where do DSBs and SSGs come from?

A

DNA-damaging agents and normal DNA replication

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17
Q

What is the purpose of recombination?

A

Necessary to re initiate DNA replication

Repairs DSBs and rectifies aborted replication process

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18
Q

What does an exonuclease do in recombination?

A

Forms a double stranded break in the recipient strand

Digests 5’ side of nick forming 3’ overhang

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19
Q

What does RecA do?

A

Coats the 3’-ssDNA overhang

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20
Q

What are recombination specific helicases?

A

RecBCD and RecQ

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21
Q

What does RecBCD do?

A

Loads RecA protein onto overhang

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22
Q

What does RecQ do?

A

Works in conjunction with RecJ exonuclease to function at single stranded gaps

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23
Q

What happens when RecA fails to assemble?

A

RecF, RecO and RecR assemble

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24
Q

What is the presynaptic filament?

A

The nucleoprotein complex formed by the assembly of contigous RecA protein on ssDNA which undergoes homology search and strand invasion

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25
What does the RuvAB complex do?
Branch migrates the crossover point
26
What does the RuvC protein do?
Holliday junction specific endonuclease which resolves the Holliday junction
27
What does the RuvABC complex do?
Recognises and cleaves the Holliday junction
28
What is oriC?
DNA replication in E.coli is origin dependent and must start from oriC
29
What is PriA?
A DNA replication protein that is required to reinitiate the replisome so Pol III can extend the nucleoprotein complex
30
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
31
What is initiation of replication?
The recognition of an origin and separation and stabilisation of parental strands
32
What is elongation?
Assembly of the replisome at the replication fork and synthesis of the daughter strands
33
What is termination?
Joining at the ends and separation of the chromosome
34
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
35
What is a primersome?
Helicase/primase complex
36
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
37
What does a DNA polymerase do?
Catalyses addition of deoxynucleotides to growing chain
38
What is a deoxynucleotide triphosphate?
Precursor to DNA polymerase which loses terminal pyrophosphate when nucleotide added
39
When is 3' - 5' proofreading exonuclease activity useful?
During bacterial replication
40
When is 5' - 3' exonuclease activity useful?
In nick translation in Pol I
41
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
42
How does proofreading work?
Incorrect nucleotide incorporated, polymerase stalls, free ss3' end forms 3' end enters exonuclease site and is cleaved to remove mismatch
43
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
44
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
45
What does Pol alpha do?
Initiates synthesis and s tightly associated with primase. Extends RNA primer by several nucleotides then dissociates
46
What does Pol delta do?
Primary DNA synthesising enzyme
47
What is PCNA?
Loads onto pol delta to make it processive so leading strand can synthesise
48
What is RFC?
The molecule that loads PCNA onto pol delta
49
How is the RNA primer removed in eukaryotes?
By nucleases FEN-1 and Rnase H1
50
What are direct repair systems?
They act directly on the damaged nucleotide to convert them back to the original form
51
How is alkylation damage reversed?
Direct transfer of alkyl group to enzyme polypeptide chain
52
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
53
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
54
Name 3 types of DNA glycolyases
Deaminated bases Oxidation products Methylated bases
55
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
56
How is the gap filled?
In E.coli DNA pol I fills it | In mammals DNA pol beta fills it
57
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
58
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)
59
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
60
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
61
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
62
What genes control the SOS response?
recA and lexA
63
What do checkpoints do?
Ensure only undamaged cells divide
64
What is RecG?
A branched DNA specific helicase that unwinds forked DNA to form four stranded Holliday junctions
65
What is DnaB?
A replicative helicase that unwinds the parental duplex ahead of the advancing replication machinery
66
What is DnaG?
A primase
67
What does PriA do in respect to RecG?
Reloads DnaB and DnaG onto the stalled replication fork DNA
68
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
69
What are the 2 possible models for stalled fork lesion repair?
Chromosome breakage and recombination | Recombination without breakage
70
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
71
Why does chromosome breakage occur?
Although lethal, it is necessary to reinitiate a blocked replication fork
72
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
73
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
74
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
75
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
76
What is Blooms syndrome?
A chromosomal disorder where there is an increases in spontaneous sister chromatid exchange leading to duplicated sequences and chromosome abberations
77
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
78
What is RecQ?
A family of helicases that can reinitiate stalled replication forks without forming a recombinant molecule
79
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
80
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
81
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
82
How does dsRNA silence genes?
By marking their mRNAs for destruction
83
What is Dicer?
An enzyme that catalyses the cleavage of dsRNA into siRNA
84
What is RISC?
RNA induced silencing complex which acts on siRNA and distinguishes between sense and antisense strands, degrading the sense strand
85
How is RISC activated?
By siRNAs unwinding by ATP
86
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
87
What do miRNAs do?
They silence genes at the protein synthesis stage by binding 3'UTR
88
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
89
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
90
What happens when the miRNA is exactly complementary?
The DNA is degraded
91
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
92
What does the splicing of introns affect?
``` Initial transcription Editing Polyadenylation of pre-mRNA Nuclear export Translational efficiency Decay of mRNA ```
93
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
94
How do introns affect RNA editing?
Required for modification of some exon sequences | A - I editing
95
What is an ADAR enzyme?
Adenine deaminase that acts on RNA | Converts adenosine to inosine in dsRNA
96
Describe the process of A - I editing
I is read as G during translation so A - I results in A - G
97
What is EJC?
The functional link between splicing and mRNA export
98
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

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