Lecture 5 DNA recombination, damage and repair

Recombination

• There are several kinds of recombination in living cells. General

recombination occurs as part of meiosis and results in crossing over

(recombination) of chromosomes.

• The Holliday model was proposed in 1964 by Robin Holliday to explain the

formation of heteroduplexes and gene conversion during meiosis. It has been

replaced by a double-strand break (DSB) model, at least for yeast and other

eukaryotes, but some of the details still hold.

• Holliday assumed that recombination is initiated by single stranded breaks in

two chromosomes, and each single strand invades the other duplex.

However, initiation probably starts with a DSB.

• A Holliday structure (joint molecule) is formed following ligation.

• Heteroduplex (DNA from each duplex) is ended by branch migration.

• The Holliday junction can be resolved in two ways: if the strands that were

originally nicked are nicked again and resealed, a region of heteroduplex

DNA is formed, but there is no recombination of flanking markers. If the other

Strands are nicked, then recombination of flanking markers occurs.

• Single-stranded DNA Single-stranded is formed by the proteins RecBCD.

• RecA binds to single-stranded DNA and promotes strand exchange with

another DNA duplex.

• RuvA and RuvB promote strand migration, and RuvC resolves the structure.

DNA Damage

• DNA damage can have endogenous (replication, metabolic byproducts [R-Mb]) or

exogenous (mutagens, carcinogens [MC]) causes.

• Oxidation, oxidative deamination and alkylation ——→ transitions/transversions.

• UV and intercalating agents ——→ frameshifts.

• Ionising radiation and/or replication errors ——→ double-stranded breaks

DNA Repair

• There are several DNA repair pathways; the most important are Direct

Reversal, Nucleotide Excision Repair (NER), Base Excision Repair (BER),

Mismatch Repair (MMR), Non-homologous End Joining (NHEJ) and

Homology Directed Repair (HDR, also called Homology Repair or HR)

• Direct reversal uses energy from sunlight to activate a photolyase enzyme

that repairs pyrimidine dimers. There is no direct reversal pathway in

mammals.

• BER requires the activity of DNA glycosylases [initiates](to remove the base without

breaking the phosphodiester backbone), and AP endonucleases, which break

the phosphodiester backbone. Some bases are removed in the 3’ to 5’

direction, and the gap is repaired by PolI (in bacteria). Most common

pathway.

• NER is used to correct bulky lesions. UvrA,B, and C cleaves at the 7th

phosphodiester bond 5’ to damage and the 3rd or 4th bond 3’ to damage. The

bases are again replaced by DNA polymerase.

• Most replication errors and short insertions/deletions are corrected by MMR.

• MutS recognises the parental (methylated) strand, binds MutL and translocates in

both directions to form a DNA loop.

• MutH makes a single-stranded nick 5’ to the unmethylated site, exonuclease

removes bases, repaired by DNA polymerase.

• Double Stranded Breaks (DSB, often caused by ionising radiation) are

repaired by NHEJ and Homology Directed Repair. NHEJ is the most

commonly used pathway in humans to repair DSBs.

• NHEJ uses “microhomologies” that are often present in single-stranded

overhangs on the ends of double-strand breaks. When the overhangs are

perfectly compatible, NHEJ usually repairs the break accurately. More often,

repair by NHEJ results in loss or gain of bases at the site of the DSB.

• In eukaryotes, during NHEJ, DSB ends are recognised by Ku70/80 proteins

and other proteins. The full details are not known, but the ends of the DNA

are processed and ligated together.

• When a homologous region of DNA is available, DSBs can be repaired by

Homology Repair. The ends of the DSBs are resected to generate protruding

3’ ends. One end invades the homologous copy. Repair then proceeds in a

very similar manner to homologous recombination.

• Xeroderma pigmentosum, Cockaynes syndrome, HNPCC and hereditary

breast cancer in humans are caused by defects in the repair enzyme

📘 FLASHCARDS (35)

Flashcard 1
Q: What is general recombination?
A: Recombination during meiosis that results in crossing over of chromosomes

Flashcard 2
Q: What is the outcome of general recombination in meiosis?
A: Exchange of genetic material between homologous chromosomes

Flashcard 3
Q: Who proposed the Holliday model and when?
A: Robin Holliday, 1964

Flashcard 4
Q: What did the Holliday model explain?
A: Heteroduplex formation and gene conversion

Flashcard 5
Q: Which model has replaced the Holliday model in eukaryotes?
A: Double Strand Break (DSB) model

Flashcard 6
Q: How did Holliday propose recombination was initiated?
A: By single-stranded breaks in both chromosomes

Flashcard 7
Q: How is recombination initiation now thought to begin?
A: With a double-stranded break

Flashcard 8
Q: What structure is formed after strand invasion and ligation?
A: Holliday junction (joint molecule)

Flashcard 9
Q: What is heteroduplex DNA?
A: DNA formed from strands originating from different duplexes

Flashcard 10
Q: What process extends heteroduplex DNA?
A: Branch migration

Flashcard 11
Q: What determines whether crossing over occurs?
A: The way the Holliday junction is resolved

Flashcard 12
Q: What happens if the originally nicked strands are cut again?
A: No recombination of flanking markers

Flashcard 13
Q: What happens if the alternative strands are cut?
A: Recombination of flanking markers occurs

Flashcard 14
Q: Which protein complex generates single-stranded DNA?
A: RecBCD

Flashcard 15
Q: What is the role of RecA?
A: Promotes strand exchange between DNA molecules

Flashcard 16
Q: Which proteins promote branch migration?
A: RuvA and RuvB

Flashcard 17
Q: Which protein resolves Holliday junctions?
A: RuvC

Flashcard 18
Q: What are endogenous causes of DNA damage?
A: Replication errors and metabolic byproducts

Flashcard 19
Q: What are exogenous causes of DNA damage?
A: Mutagens and carcinogens

Flashcard 20
Q: Which types of damage cause transitions or transversions?
A: Oxidation, deamination, alkylation

Flashcard 21
Q: Which agents cause frameshift mutations?
A: UV light and intercalating agents

Flashcard 22
Q: What commonly causes double-stranded breaks?
A: Ionising radiation and replication errors

Flashcard 23
Q: What is the most common DNA repair pathway?
A: Base Excision Repair (BER)

Flashcard 24
Q: Which enzymes initiate BER?
A: DNA glycosylases

Flashcard 25
Q: Which enzyme cuts the backbone in BER?
A: AP endonuclease

Flashcard 26
Q: Which polymerase fills gaps during BER in bacteria?
A: DNA polymerase I

Flashcard 27
Q: What type of damage is repaired by NER?
A: Bulky lesions

Flashcard 28
Q: Which proteins function in bacterial NER?
A: UvrA, UvrB, and UvrC

Flashcard 29
Q: What does mismatch repair correct?
A: Replication errors and small insertions/deletions

Flashcard 30
Q: Which protein recognizes methylated DNA in MMR?
A: MutS

Flashcard 31
Q: Which enzyme creates the nick during MMR?
A: MutH

Flashcard 32
Q: What are the two main DSB repair pathways?
A: NHEJ and Homology Directed Repair (HDR)

Flashcard 33
Q: Which DSB repair pathway is most common in humans?
A: Non-homologous end joining (NHEJ)

Flashcard 34
Q: Which proteins recognize DSBs during NHEJ?
A: Ku70/Ku80

Flashcard 35
Q: Name one human disease caused by DNA repair defects.
A: Xeroderma pigmentosum (also Cockayne syndrome, HNPCC, hereditary breast cancer)

🧪 MULTIPLE CHOICE QUESTIONS (35)

MCQs

General recombination occurs during:
A. Mitosis
B. Meiosis
C. Transcription
D. Translation
Crossing over results in:
A. DNA repair
B. Gene silencing
C. Exchange of chromosome segments
D. Mutation
The Holliday model explains:
A. DNA replication
B. Transcription
C. Heteroduplex formation
D. Telomere extension
The Holliday model was proposed in:
A. 1953
B. 1964
C. 1972
D. 1980
Modern models suggest recombination begins with:
A. Single-strand breaks
B. Base damage
C. Double-strand breaks
D. Frameshifts
A Holliday junction is a:
A. Protein complex
B. Joint DNA molecule
C. Repair enzyme
D. RNA structure
Branch migration results in:
A. Strand degradation
B. DNA synthesis
C. Extension of heteroduplex DNA
D. Mutation
Resolution of Holliday junctions determines:
A. Mutation rate
B. Whether crossing over occurs
C. DNA replication speed
D. Protein synthesis
Which protein creates single-stranded DNA?
A. RecA
B. RuvC
C. RecBCD
D. MutS
RecA promotes:
A. DNA cleavage
B. Strand exchange
C. Primer synthesis
D. Ligation
RuvA and RuvB are involved in:
A. DNA unwinding
B. Branch migration
C. Base removal
D. Ligation
RuvC functions to:
A. Bind DNA
B. Resolve Holliday junctions
C. Add nucleotides
D. Repair mismatches
Endogenous DNA damage can arise from:
A. UV radiation
B. Ionizing radiation
C. Replication errors
D. Intercalating agents
Transitions and transversions are caused by:
A. UV damage
B. Alkylation
C. Frameshifts
D. Strand breaks
Frameshift mutations are often caused by:
A. Oxidation
B. Alkylation
C. UV and intercalating agents
D. Deamination
Ionising radiation commonly causes:
A. Point mutations
B. Frameshifts
C. Double-stranded breaks
D. Methylation
Which repair pathway uses photolyase?
A. BER
B. NER
C. Direct reversal
D. MMR
Why does direct reversal not occur in mammals?
A. No photolyase enzyme
B. No UV damage
C. No pyrimidines
D. No ATP
BER begins with the action of:
A. DNA polymerase
B. DNA ligase
C. DNA glycosylase
D. RNase
Which enzyme cleaves the backbone in BER?
A. Ligase
B. AP endonuclease
C. Exonuclease
D. Helicase
NER primarily repairs:
A. Single base damage
B. Bulky lesions
C. Mismatches
D. Strand breaks
Which proteins act in bacterial NER?
A. MutS, MutL
B. RecA, RecBCD
C. UvrA, UvrB, UvrC
D. Ku70/80
Mismatch repair corrects:
A. UV damage
B. Bulky adducts
C. Replication errors
D. Double-strand breaks
Which protein recognizes methylated DNA?
A. MutH
B. MutS
C. MutL
D. RecA
MutH creates a:
A. Double-strand break
B. Nick in the unmethylated strand
C. Primer
D. Holliday junction
Which enzyme fills the gap after MMR excision?
A. Ligase
B. DNA polymerase
C. Helicase
D. Photolyase
DSBs are repaired by:
A. BER only
B. NER only
C. NHEJ and HDR
D. MMR
Most DSB repair in humans uses:
A. HDR
B. BER
C. NHEJ
D. Direct reversal
NHEJ often results in:
A. Perfect repair
B. Base loss or gain
C. No repair
D. Telomere addition
Microhomologies are used in:
A. HDR
B. BER
C. NHEJ
D. MMR
Ku70/80 proteins function in:
A. BER
B. HDR
C. NHEJ
D. MMR
Homology-directed repair requires:
A. No template
B. Homologous DNA
C. RNA
D. Photolyase
HDR repair begins with:
A. Ligation
B. Strand invasion
C. End resection producing 3′ overhangs
D. Base excision
Xeroderma pigmentosum is caused by defects in:
A. Replication enzymes
B. Repair enzymes
C. Translation factors
D. Transcription factors
Hereditary breast cancer is linked to defects in:
A. DNA repair pathways
B. RNA processing
C. Protein folding
D. Metabolism