tony weiss series Flashcards
1
Q
• Homologous recombination
A
• Homologous recombination is essential in meiosis for
generating diversity and for chromosome segregation,
and in mitosis to repair DNA damage and stalled
replication forks.
2
Q
• Site-specific recombination
A
• Site-specific recombination involves specific DNA
sequences.
3
Q
• somatic recombination
A
• somatic recombination – Recombination that occurs in
nongerm cells (i.e., it does not occur during meiosis);
most commonly used to refer to recombination in the
immune system.
• Recombination systems have been adapted for
experimental use.
4
Q
Homologous Recombination Occurs
between Synapsed Chromosomes in Meiosis
A
• Chromosomes must synapse (pair) in order for chiasmata to form where crossing-over occurs. • The stages of meiosis can be correlated with the molecular events at the DNA level.
5
Q
• sister chromatid
A
– Each of two identical copies
of a replicated chromosome; this term is used as
long as the two copies remain linked at the
centromere.
– Sister chromatids separate during anaphase in
mitosis or anaphase II in meiosis.
6
Q
bivalent –
A
• The structure containing all four
chromatids (two representing each homolog) at
the start of meiosis.
7
Q
• synaptonemal complex –
A
The morphological structure
of synapsed chromosomes.
8
Q
• joint molecule –
A
A pair of DNA duplexes that are
connected together through a reciprocal exchange of
genetic material.
9
Q
• The double-strand break repair (DSBR)
A
• The double-strand break repair (DSBR) model of
recombination is initiated by making a double-strand
break in one (recipient) DNA duplex and is relevant for
meiotic and mitotic homologous recombination.
• In 5’ end resection, exonuclease action generates 3′–
single-stranded ends that invade the other (donor)
duplex.
10
Q
Double-Strand Breaks
Initiate Recombination
A
• When a single strand from one duplex displaces its counterpart in the other duplex (single-strand invasion), it creates a branched structure called a D loop. • Strand exchange generates a stretch of heteroduplex DNA consisting of one strand from each parent. • New DNA synthesis replaces the material that has been degraded. • branch migration – The ability of a DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous.
11
Q
• branch migration –
A
The ability of a DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous.
12
Q
Holliday junctions.
A
Capture of the second DSB end by annealing generates
a recombinant joint molecule in which the two DNA
duplexes are connected by heteroduplex DNA and two
Holliday junctions.
13
Q
Double-Strand Breaks Initiate
Recombination
A
• Capture of the second DSB end by annealing generates
a recombinant joint molecule in which the two DNA
duplexes are connected by heteroduplex DNA and two
Holliday junctions.
• The joint molecule is resolved into two separate duplex
molecules by nicking two of the connecting strands.
• Whether recombinants are formed depends on whether
the strands involved in the original exchange or the other
pair of strands are nicked during resolution.
14
Q
Gene Conversion Accounts for
Interallelic Recombination
A
• Heteroduplex DNA that is created by recombination can
have mismatched sequences where the recombining
alleles are not identical.
• Repair systems may remove mismatches by changing
one of the strands so its sequence is complementary to
the other.
15
Q
Gene Conversion Accounts for
Interallelic Recombination
A
• Mismatch (gap) repair of heteroduplex DNA generates nonreciprocal recombinant products called gene conversions.
16
Q
Dependent Strand-
Annealing Model
A
15.5 The Synthesis-
• The synthesis-dependent strand-annealing model (SDSA) is relevant for mitotic recombination, as it produces gene conversions from double-strand breaks without associated crossovers.
17
Q
What happens when
recombination goes wrong?
A
Mutations in BLM, which encodes a RecQ helicase, give rise to Bloom’s syndrome,
a disorder associated with cancer predisposition and genomic instability.
• A defining feature of Bloom’s syndrome is an elevated frequency of sister chromatid
exchanges
18
Q
The Single-Strand Annealing Mechanism Functions at Some Double-Strand Breaks
A
Single-strand annealing (SSA) occurs at double- strand breaks between direct repeats.
19
Q
TheSingle-StrandAnnealingMechanism Functions at Some Double-Strand Breaks
A
- Resection of double-strand break ends results in 3′ single-stranded tails.
- Complementarity between the repeats allows for annealing of the single strands.
- The sequence between the direct repeats is deleted after SSA is completed.
20
Q
Break-InducedReplicationCanRepair Double-Strand Breaks
A
- Break-induced replication (BIR) is initiated by a one-ended double-strand break.
- BIR at repeated sequences can result in translocations.
21
Q
PairingandSynaptonemalComplex Formation Are Independent
A
• Mutations can occur in either chromosome pairing or synaptonemal complex formation without affecting the other process.
22
Q
The Bacterial RecBCD System Is Stimulated by chi Sequences
A
- The RecBCD complex has nuclease and helicase activities.
- RecBCD binds to DNA downstream of a chi sequence, unwinds the duplex, and degrades one strand from 3′–5′ as it moves to the chi site.
- The chi site triggers loss of the RecD subunit and nuclease activity.
23
Q
Strand-TransferProteinsCatalyze Single-Strand Assimilation
A
- RecA forms filaments with single-stranded or duplex DNA and catalyzes the ability of a single-stranded DNA with a free 3′ to displace its counterpart in a DNA duplex.
- presynaptic filaments – Single-stranded DNA bound in a helical nucleoprotein filament with a strand transfer protein such as Rad51 or RecA.
24
Q
presynaptic filaments
A
• – Single-stranded DNA bound in a helical nucleoprotein filament with a strand transfer protein such as Rad51 or RecA.
25
HollidayJunctionsMustBeResolved
• The bacterial Ruv complex acts on recombinant junctions.
• RuvA recognizes the structure of the junction and RuvB is a helicase that catalyzes branch migration.
A model for the interaction of RuvA with its DNA target using a simple model of a Holliday junction in which the double- stranded B-DNA arms are held in a square-planar arrangement.
26
HollidayJunctions Must Be Resolved
• RuvC cleaves junctions to generate recombination intermediates.
• Resolution in eukaryotes is less well understood, but a number of meiotic and mitotic proteins are implicated.
simple model of a RuvA/RuvB/DNA complex.
RuvA binds the Holliday junction at the central crossover point and targets two RuvB hexamers onto opposite arms of the DNA where they encircle the DNA duplexes and facilitate branch migration in concert with RuvA in an ATP dependent manner.
27
photoreactivation
– A repair mechanism that uses a white light-dependent enzyme to split cyclobutane pyrimidine dimers formed by ultraviolet light.
28
• excision repair
– A type of repair system in which one
| strand of DNA is directly excised and then replaced by resynthesis using the complementary strand as template.
29
• base excision repair (BER)
– A pathway of excision repair that recognizes damage to single bases, such as deamination or alkylation, and either repairs the base alone (short-patch repair) or replaces 2–10 nucleotides (long-patch repair).
30
• nucleotideexcisionrepair(NER)–
Anexcision repair pathway that recognizes bulky lesions in DNA (such as UV-induced pyrimidine dimers)
31
Repair Systems Correct Damage to DNA
* Recombination-repair systems use recombination to replace the double- stranded region that has been damaged.
* All these systems are prone to introducing errors during the repair process.
* Photoreactivation is a nonmutagenic repair system that acts specifically on pyrimidine dimers.
32
Excision Repair Systems in E. coli
* The Uvr system makes incisions ~12 bases apart on both sides of damaged DNA, removes the DNA between them (excision), and resynthesizes new DNA.
* Transcribed genes are preferentially repaired when DNA damage occurs.
33
Repair Systems Correct Damage to DNA
Repair systems recognize DNA sequences that do not conform to standard base pairs.
Excision systems remove one strand of DNA at the site of damage and then replace it.
34
BaseExcisionRepairSystems Require Glycosylases
* Base excision repair is triggered by directly removing a damaged base from DNA.
* Base removal triggers the removal and replacement of a stretch of polynucleotides.
* The nature of the base removal reaction determines which of two pathways for excision repair is activated.
* The polδ/ε pathway replaces a long polynucleotide stretch; the polβ pathway replaces a short stretch.
35
BaseExcisionRepairSystems Require Glycosylases
* Uracil and alkylated bases are recognized by glycosylases and removed directly from DNA.
* Glycosylases and photolyase (a lyase) act by flipping the base out of the double helix, where, depending on the reaction, it is either removed or modified and returned to the helix.
36
DNA is fragile
* Excessive exposure to UV radiation e.g. sunlight
* Environment e.g. tobacco smoke
* Oxidative damage from by-products of metabolism e.g. free radicals.
* An individual cell can suffer up to one million DNA changes per day
37
Error-Prone Repair
* Damaged DNA that has not been repaired causes DNA polymerase III to stall during replication.
* DNA polymerase V (coded by umuCD) or DNA polymerase IV (coded by dinB) can synthesize a complement to the damaged strand.
* The DNA synthesized by repair DNA polymerases often has errors in its sequence (error-prone synthesis).
38
Controlling the Direction of Mismatch Repair
• mutator – A mutation or a mutated gene that increases the basal level of mutation.
– Such genes often code for proteins that are involved in repairing damaged DNA.
• The mut genes code for a mismatch repair system that deals with mismatched base pairs.
39
• mutator
– A mutation or a mutated gene that increases the basal level of mutation.
40
Controlling the Direction of Mismatch Repair
* There is a bias in the selection of which strand to replace at mismatches.
* The strand lacking methylation at a hemimethylated GATCCTAG is usually replaced.
* The mismatch repair system is used to remove errors in a newly synthesized strand of DNA. At G-T and C-T mismatches, the T is preferentially removed.
41
Controlling the Direction of Mismatch Repair e.g.
Examples of deamination which involves the removal of an amino group.
Accidental deamination may change the cytosine to uracil, or the methylated cytosine to thymine.
42
Controlling the Direction of Mismatch Repair
* There is a bias in the selection of which strand to replace at mismatches.
* The strand lacking methylation at a hemimethylated GATCCTAG is usually replaced.
* The mismatch repair system is used to remove errors in a newly synthesized strand of DNA. At G-T and C-T mismatches, the T is preferentially removed.
43
Controlling the Direction of Mismatch Repair
•
| Eukaryotic MutS/L systems repair mismatches and insertion/deletion loops.
44
Recombination-Repair Systems in
| E. coli
• The rec genes of E. coli code for the principal
recombination-repair system.
• The recombination-repair system functions when replication leaves a gap in a newly synthesized strand that is opposite a damaged sequence.
45
Recombination-Repair Systems in
| E. coli
* The single strand of another duplex is used to replace the gap (single-strand exchange).
* The damaged sequence is then removed and resynthesized.
46
Recombination Is an Important Mechanism to Recover from Replication Errors
* A replication fork may stall when it encounters a damaged site or a nick in DNA.
* A stalled fork may reverse by pairing between the two newly synthesized strands.
47
Recombination Is an Important Mechanism to Recover from Replication Errors
•
A stalled fork may restart after repairing the damage and use a helicase to move the fork forward.
• The structure of the stalled fork is the same as a Holliday junction and may be converted to a duplex and DSB by resolvases.
48
Nonhomologous End-Joining Also Repairs Double-Strand Breaks
* The nonhomologous end- joining (NHEJ) pathway can ligate blunt ends of duplex DNA.
* Mutations in double-strand break repair pathways cause human diseases.
49
DNA Repair in Eukaryotes Occurs in the Context of Chromatin
* Different patterns of histone modifications may distinguish stages of repair or different pathways of repair.
* Remodelers and chaperones are required to reset chromatin structure after completion of repair.
50
DNA Repair in Eukaryotes Occurs in the Context of Chromatin
* Both histone modification and chromatin remodeling are essential for repair of DNA damage in chromatin.
* H2A phosphorylation is a conserved double-strand break-dependent modification that recruits chromatin modifying activities and facilitates assembly of repair factors.
51
For example:
Li–Fraumeni syndrome is an extremely rare autosomal dominant hereditary disorder.
It greatly increases susceptibility to cancer. This syndrome is also known as the Sarcoma, breast, leukaemia and adrenal gland (SBLA) syndrome.
Disorders highlighted in grey are those that exhibit defects in several responses to DNA damage (e.g. repair, checkpoint activation). XP; xeroderma pigmentosum, XP-A; XP complementation group A, CS; Cockayne syndrome, LFS; Li–Fraumeni syndrome, LIG4; DNA ligase IV syndrome, XLF/Cernnunos-SCID; severe combined immunodeficiency caused by mutations in XLF (XRCC4-like factor)/Cernnunos, A-T; Ataxia telangiectasia.
52
transposon (transposable element) –
A DNA sequence able to insert itself (or a copy of itself) at a new location in the genome without having any sequence relationship with the target locus.
53
retrovirus –
An RNA virus with the ability to convert its sequence into DNA by reverse transcription.
54
retrotransposon(retroposon)
–A transposon that mobilizes via an RNA form; the DNA element is transcribed into RNA, and then reverse-transcribed into DNA, which is inserted at a new site in the genome. It does not have an infective (viral) form.
– Commonly, retrotransposons containing long terminal repeats (LTRs) are referred to as retrotransposons, while non-LTR-containing retrotransposons are referred to as retroposons.
55
insertion sequence
An insertion sequence (IS) is a transposon that codes for the enzyme(s) needed for transposition flanked by short inverted terminal repeats.
The target site at which an insertion sequence is inserted is duplicated during the insertion process to form two repeats in direct orientation at the ends of the transposon (direct repeats).
56
Transposition Occurs by Both Replicative and Nonreplicative Mechanisms
• Most transposons use a common mechanism in which staggered nicks are made in target DNA, the transposon is joined to the protruding ends, and the gaps are filled.
57
Insertion Sequences Are Simple Transposition Modules
* The length of the direct repeat is 5 to 9 bp and is characteristic for any particular insertion sequence.
* transposase – The enzyme activity involved in insertion of transposon at a new site.
58
Transposition Occurs by Both Replicative and Nonreplicative Mechanisms
The order of events and exact nature of the connections between transposon and target DNA determine whether transposition is replicative or nonreplicative.
59
Transposition Occurs by Both Replicative and Nonreplicative Mechanisms
* resolvase – The enzyme activity involved in site-specific recombination between two copies of a transposon that has been duplicated.
* composite transposons – Transposable elements consisting of two IS elements (which can be the same or different) and the DNA sequences between the IS elements; the non-IS sequences often include gene(s) conferring antibiotic resistance.
60
• resolvase –
The enzyme activity involved in site-specific recombination between two copies of a transposon that has been duplicated.
61
• composite transposons –
Transposable elements consisting of two IS elements (which can be the same or different) and the DNA sequences between the IS elements; the non-IS sequences often include gene(s) conferring antibiotic resistance.
62
Transposons Cause Rearrangement of DNA
* Homologous recombination between multiple copies of a transposon causes rearrangement of host DNA.
* Homologous recombination between the repeats of a transposon may lead to precise or imprecise excision.
63
Replicative Transposition Proceeds through a Cointegrate
• Replication of a strand transfer complex generates a cointegrate, which is a fusion of the donor and target replicons.
• The cointegrate has two copies of the transposon, which lie between the original replicons
• Recombination between the transposon copies regenerates the original replicons, but the recipient has gained a copy of the transposon.
• The recombination reaction (resolution) is catalyzed by a resolvase coded by the transposon..
64
Nonreplicative Transposition Proceeds by Breakage and Reunion
* Nonreplicative transposition results if a crossover structure is nicked on the unbroken pair of donor strands and the target strands on either side of the transposon are ligated.
* Two pathways for nonreplicative transposition differ according to whether the first pair of transposon stands are joined to the target before the second pair are cut (Tn5), or whether all four strands are cut before joining to the target (Tn10).
65
Retrotransposons
* LTR retrotransposons mobilize via an RNA that is similar to retroviral RNA, but does not form an infectious particle.
* long-interspersed nuclear elements (LINEs) – A major class of retrotransposons that occupy ~21% of the human genome.
66
LINEs Use an Endonuclease to Generate a Priming End
• LINES do not have LTRs and require the retroposon to code for an endonuclease that generates a nick to prime reverse transcription.
67
Alu Family: Many Widely Dispersed Members
• A major part of repetitive DNA in mammalian genomes consists of repeats of a single family organized like transposons and derived from RNA polymerase III transcripts.
68
Alu-associated genome rearrangement
• “The ACE gene, encoding angiotensin-converting enzyme, has 2 common variants, one with an Alu insertion (ACE-I) and one with the Alu deleted (ACE-D). This variation has been linked to changes in sporting ability: the presence of the Alu element is associated with better performance in endurance-oriented events (e.g. triathlons), whereas its absence is associated with strength- and power-oriented performance”
there are over one million Alu sequences interspersed throughout the human genome, and it is estimated that about 10.7% of the human genome consists of Alu sequences.
69
Xeroderma pigmentosum (XP)
.Xeroderma pigmentosum, or XP:
• autosomal recessive genetic disorder of DNA
repair
• reduced ability to repair damage caused by
ultraviolet light
• in extreme cases, all exposure to sunlight is
forbidden, individuals are often colloquially
referred to as Children of the Night
• most common defect in XP is nucleotide
excision repair (NER) enzymes are mutated
• leads to a reduction in or elimination of NER
70
BRCA1 and BRCA2
Women with abnormal BRCA1 or BRCA2 gene:
• up to 80% risk of developing breast cancer by
age 90 (men at elevated risk too)
• increased risk of developing ovarian cancer:
~55% for women with BRCA1 mutations and
about 25% for women with BRCA2 mutations
71
Specialized Recombination Involves
| Specific Sites
```
Bacteriophage
lambda lacks tail
fibres, but attaches
to a host cell via
molecules at the
base of the tail.
```
```
Bacteriophage
lambda is a
lysogenic virus that
can incorporate its
dsDNA with that of
its host cell, E. coli.
```
72
Specialized Recombination Involves
| Specific Sites
```
• Specialized recombination
involves reaction between
specific sites that are not
necessarily homologous.
• recombinase – Enzyme that
catalyzes site-specific
recombination.
• Phage lambda integrates into
the bacterial chromosome by
recombination between a site
on the phage and the att site
on the E. coli chromosome
```
73
• core sequence –
The segment of DNA that is
common to the attachment sites on both the
phage lambda and bacterial genomes.
– It is the location of the recombination event that
allows phage lambda to integrate.
74
Specialized Recombination Involves
| Specific Sites
• core sequence – The segment of DNA that is
common to the attachment sites on both the
phage lambda and bacterial genomes.
– It is the location of the recombination event that
allows phage lambda to integrate.
• The phage is excised from the chromosome by
recombination between the sites at the end of
the linear prophage.
• Phage lambda int codes for an integrase that
catalyzes the integration reaction.
75
Site-Specific Recombination Involves
| Breakage and Reunion
• Cleavages staggered by 7 bp are made in both attB and
| attP and the ends are joined crosswise.
76
Cre and Lox
* Cre and Lox are experimentally derived from P1
| * P1 is a phage that infects E. coli
77
• Cre:
– a site-specific recombinase
| – DNA recombination on target DNA flanked with loxP sites
78
Site-Specific Recombination
| Resembles Topoisomerase Activity
• Two enzyme units bind to each recombination site and
the two dimers synapse to form a complex in which the
transfer reactions occur.
79
Recombination Pathways Adapted for
| Experimental Systems
• Mitotic homologous recombination allows for targeted
transformation.
• The Cre/lox system allows for targeted recombination
and gene knockout construction.
80
Cre and Lox methodology
| is not limited to mice
A common tumor type is induced in fish that are made transgenic for the
oncogene RAS.
(A)Normal zebrafish side view.
(B) Transgenic fish (46 dpf) with externally visible tumour mass at the tail region.
