Replication, Repair and Recombination L7-9

Question 1

What base is most susceptible to spontaneous hydrolytic damage?
This is the most common damage in cells

Answer 1

guanine

5000 times a day

Question 2

What does the deamination of cytosine make?

Answer 2

Uracil
100 times a a day
Not a natural base in DNA - identified as damage by repair systems

Question 3

What does the deamination of 5-me-cytosine make?

5mC only exists in higher Euks

Answer 3

Thymidine

Will be a G-T base pair (no way of identifying which was the original strand)

Question 4

What does guanine change to during spontaneous oxidative damage?

Answer 4

8-oxoguanine
unusual base structure
Following replication can result in a mismatch pairing with ADENINE

Question 5

What does guanine change to during chemical induced damage-alkylation?

Answer 5

O^6methylguanine
unusual base structure recognised by repair systems
Alkylating agents are reactive compounds that can transger methyl or ethyl groups to DNA base
O^6methylguanine base pairs with THYMINE

Question 6

What does guanine change to during chemical induced damage-carcinogens?

Answer 6

Guanine benzopyrene
Bulky heterocyclic ring
Intercalates and BENDS DNA-replication and transcription halted

Question 7

What bases does UV 200-300nm damage occur at?

Answer 7

Adjacent thymidines or adjacent cyteines on the same strand.
PYRIMIDINE DIMERS
Creates CYCLOBUTANE due to excitation from the UV light
Blocks/stalls replication and transcription

Question 8

What are examples of accidental and programmed damage?

Answer 8

ACCIDENTAL
ionizing radiation/DNA-damaging agents/desiccation (bacteria can produce spores that induce ds breaks)/ inappropriate nuclease activity/replication past a DNA nick
PROGRAMMED
Meiotic recombination/VDJ recombination

Question 9

What do DNA photolyases repair and what co-factor do they have?

Widely distributed in Proks and Euks but not in HUMANS.

Answer 9

Pyrimidine dimers
Flavoprotein-All use anionic FADH- as a redox active cofactor to break the cyclobutane ring. Also have light harvesting co-factors that act like antenna to help collect more photons to the flavin co-factor to drive the reaction.
Light/photoreactivation is used to break the ring structure.
Position flavin co-factor next to the damaged base.
Reduced form of FADH- supplies electrons to the ring to allow it to break and accepts electrons once the reaction has occurred.

Question 10

What enzyme has evolved to repair O6methylguanine (chemical induced damage-alkylation)?

Answer 10

DNA Alkyltansferases
O6methylguanine methyltansferases
CATALYTIC CYSTEINE acts as acceptor for the alkylating group-enzyme becomes alkylated
Suicide reaction-doesnt turnover like a classical enzyme and must be degraded

Question 11

What enzymes are involved in base excision repair?

Answer 11

(BASE) DNA glycosylases
cuts the N-glycosidic linkage b/w the sugar and the base and leaves an abasic site and an OH group
AP endonucleases and exonucleases/phophodiesterases remove sugar phosphate-cuts the phosphodiester bond and the 3’OH on the sugar residue.
Further enzymes remove this sugar residue from the DNA to leave a gap which can be copied by DNA pols. 3’OH left exposed to be copied by DNA pol I

Question 12

BASE EXCISION REPAIR enzymes:
Deamination
Alkylation
UV radiation
oxidation
ALL FORM AP SITE

Answer 12

Deamination=Uracil DNA glycosylase (UDG)/mismatch specific DNA glycosylase (MUG)
Alkylation=AlkA
UV radiation=Endo V
oxidation=MutY/Endo III

Question 13

How many DNA glycosylases are there in humans?

Answer 13

11

Different sorts of damage recognised by different glycosylases

Question 14

What enzymes deal with oxidised guanine (8-oxoguanine)?

Base excision repair

Answer 14

MutT,M,Y

Question 15

What does MutT do?

Answer 15

Hydrolyses 8-oxoguanine (removes the ti-phosphates) in the pool of NTPs so that it can’t be used by the DNA pol.
Damage of guanine can occur in the NT pool rather than when it is on the DNA.

Question 16

What does MutM do?

Answer 16

Acts as a glycosylase and removes G=O when 8-oxoguanine is paired with C to create an abasic site where the DNA is naturally repaired back to G=C

Question 17

What does MutY do?

Answer 17

MutY acts as a glycosylase and removes A that is paired with G=O since 8-oxoguanine pairs with adenine AFTER REPLICATION. After C is inserted into the abasic site MutM can then remove the G=O.

If MutM repaired here then you would get AT not GC.
MutY only flips out A if its paired with a G=O

Question 18

What is NUCLEOTIDE EXCISION REPAIR used for?

Answer 18

Versatile recognition of bulky damage. e.g. pyrimidine dimers
Enzymes=UvrA,B,C (plus D helicase)
These can bind together upon binding ATP and hydrolyse ATP as they bind to the DNA (ATP-dependent damage location by UvrA/B)
DNA damage distorts the structure and causes extra flexibility in the DNA. The complex binds and bends the DNA (ATP hydrolyses)
Further ATP hydrolysis releases the UvrA (damage verification UvrA dissociates)
UvrB unwinds the section of DNA around damage and UvrC binds to UvrB only when UvrA is not present (irreversibly locked onto the repair pathway)

Damaged is removed-glycosylases flip out the base and cut.

Question 19

Where does UvrC cut in Nucleotide excision repair?

Answer 19

UvrC cleaves the 4th-5th bond 3’ to lesion
and cleaves the 8th bond 5’ to lesion

UvrD helicase displaces the 12-13nt region
gap filling by DNA pol I, ligase REPAIR

Question 20

What repair pathway is not used by E.coli (yet some bacteria use it)?

Answer 20

NHEJ

Question 21

What enzymes are involved in NHEJ?
Can repair double end breaks ONLY

HR can repair both single and double stranded breaks.

Answer 21

Ku70/80 (one of the most abundant proteins in human cells)
DNA-PKcs
Artemis (5’-3’ exo/endo activity)
Ligase IV, Cernunnos-XLF XRCC4

Question 22

What phase of the cell cycle does HR occur?

Answer 22

S phase

Since here you have multiple copies of DNA.

Question 23

What are the proteins involved in HR in E.coli?

Answer 23

RecBCD-processing DNA ends to produce recombinagenic 3’ end and ensemble of strand exchange filament
RecA-Homology search and strand exchange (ATPase)
RuvAB-Holliday junction branch migration (RuvA is a tetramer)
RuvC-Holliday junction resolution/cleavage (dimer)

Question 24

HR RecBCD what are all the individual components?
RecBCD binds to the DNA ends not internal sites
This enzyme produces the 3’ overhang for HR

Answer 24

RecB- 3’-5’ helicase + endoNuclease/RecA loading
RecC- DNA recognition
RecD- 5’-3’ helicase (ATPase that unwinds the dsDNA/hydrolysis of ATP)

Question 25

What is the Chi site?

Answer 25

GCTGGTGG
site that RecBCD recognises and changes conf so that the nuclease no longer chews up the 3’ end - leaving a 3’ overhang.
Recombination occurs near chi site. The freq decreases the further away from the chi site

Question 26

RecA

Answer 26

Forms right handed nucleoprotein filaments on ss DNA
DNA extended 1.5 fold-DNA stretched to bind within this filament
Loaded onto DNA by RecBCD.
REQUIREMENTS
1) sequence complementarity
2) region of ssDNA (3’OH normally produced)
3) DNA ends required (to allow strands to intertwine)
RecA filament has catalytic sites for strand exchange

Question 27

What protein induces programmed dsDNA breaks in yeast during mieosis?

Answer 27

Spo11 (nuclease)
Dimeric protein with tyrosine residue that provides an OH group to act as a nucleophile to cut the DNA. Protein becomes covalently bound to the DNA.
Cuts DNA in hot spots where the DNA is less tightly packaged (DNA is more accessible to nuclease)
Some cuts can be reversed after meiosis is finished.