Genetics Exam 3

direct repair

photolyase uses light energy to break crosslinks in thymine dimers

excision repair

recognition enzyme sees damage, then the region of damaged bases is excised, and new DNA synthesis follows

base excision

uracil glycosylase removes the base directly followed by replacement of a single base of short stretch by DNA pol I

nucleotide excision

recognizes damage, creates incision, excises region, synthesizes new strand, ligase seals the nick

mismatch excision

corrected by distinguishing between old and new strands and using DNA pol III

recombination repair

uses homologous recombination to get undamaged original copy that replaces the damaged one

recombination-repair-retrieval system

corrects replication mistakes by recombining with a good copy of the damaged region

nonhomologous end joining

fixes double stranded breaks created by UV or antibody shuffling

error prone systems

tolerance systems allow replication in case of structural damage accepting high error rate

deamination

removing an amino group (cytosine → uracil) OR (5-methyl-cytosine → thymine)

how is deamination repaired?

base excision repair

thymine dimer formation

when two adjacent thymines covalently link and distort the duplex

how are thymine dimer duplexes repaired?

excision

alkylation of bases

adding a methyl group to a guanine to form methyl-guanine

how is alkylation of bases repaired?

dealkylation

depurination

removing a purine (adenine or guanine)

how is depurination repaired?

base replacement (insertion)

what is the connection of DNA repair with gene transcription in eukaryotes?

RNA pol

what protein serves as a link between DNA repair and gene transcription in bacteria?

Mfd

What are two major NER pathways in eukaryotes?

Global genome repair and transcription-linked

Global genome repair

XPC scans the DNA for a thymine dimer, XPG acts as an endonuclease to cut on either side of the dimer, and pol delta or epsilon fill in the gap (\~25-30 nt)

transcription linked

RNA pol II is at a thymine dimer, the core of TFIIH (helicase XPD) recognizes the stalled RNA pol II and binds, and then the RNA pol II subunit is flagged for polyubiquitination so a protease degrades the RNA pol II, and pol delta or epsilon fill in the gap (\~25-30 nt)

What protein determines which pathway is chosen in eukaryotic BER?

APE1

What signal activates RecA?

Lot of DNA damage

How does RecA affect its targets?

RecA causes autoproteolysis of UmuD to make is UmuD’, which is the activated form of DNA pol V

Which types of mutation do stop transcription/replication?

thymine dimer by UV, alkylation(methylation) of G, single-stranded nick, and removal of base(depurination)

Which types of mutation do not stop transcription/replication?

misincorporation and deamination

Incision

endonuclease cleavage on both sides of damaged base

Excision

5’ → 3’ exonuclease removes DNA between nicks

Uvr short patch

removes about 12 nt total; 99% of the systems

Uvr long patch

removes 1500-9000 nt; 1% of the systems

Components of Uvr system, steps in mechanisms, and when is ATP used?

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1. Encoded by 3 genes urvA,B,C
2. UvrA recognizes distorted DNA as a dimer with UvrB, UvrA dissociates(requires ATP)
3. UvrB recruits UvrC. UvrBC cuts (requires ATP) DNA from both the 5’ and 3’ side of the damaged base
4. UvrD(helicase) unwinds the cut region to release the strand (requires ATP)
5. DNA pol (I/II/III) synthesizes the new DNA strand
6. Ligase seals the nick

UvrA

recognizes distorted DNA

UvrB

recruits UvrC; nicks

UvrC

nicks

UvrD

helicase

Which DNA polymerase fills in the Uvr-generated gap?

DNA pol I

Mfd protein

A link between transcription and DNA repair by binding to stalled RNA pol and displacing it. It then recruits UvrAB proteins and directs the repair of the template strand

TFIIH

Helicase; opens up the DNA for transcription

XPG

Cuts DNA on either side of damage in eurkaryotes (NER); endonuclease; related to FEN1

XPD

used in nucleotide excision repair; same ad Rad3 in yeasts

XPB

helicase

Rad4

Recognizes damaged DNA and then recruits repair enzymes; flips AA, leaving damaged TT exposed

XpC

human form of yeast Rad4

Which DNA polymerase(s) fill-in the gap left in eukaryotes by NER?

The replication enzyme (DNA pol delta or epsilon)

BER: role of glycosylases?

removes the base

BER: role of lyase?

nicks the DNA chain after the base has been removed and the deoxyribose ring has been opened (some glycosylases are also lyases)

APE1

Either recruits pol delta/epsilon or pol beta

Long patch pathway

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* Glycosylase cuts out a base
* APE1 binds and acts as an endonuclease to cut the phosphodiester backbone
* APE1 recruits pol delta/epsilon and a fill in reaction occurs to displace a few bases in front of it because it has no nick translation
* FEN1 cuts off the flap of extra bases
* Ligation occurs to seal the new \~2-10 nt

short patch repair

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* Glycosylase cuts out a base
* Lyase nicks the phosphodiester backbone
* APE1 is recruited to the nick and recruits pol beta
* Pol beta replaces 1 nucleotide
* Ligase seals

base flipping

used by methylase and glycosylase

Role of DNA pol IV and V

They can synthesize a complement to the damaged strand = error prone activity (by using a by-pass mechanism with RecA)

Role of DNA pol II

repair enzyme when the cell is not under high error prone conditions

UmuDC

Codes DNA pol V

Role of RecA in activating DNA pol IV

recognizes when the cell is in heavy mutagenesis and it changes conformation to bind to specific target proteins so that it can put a kink in the protein at a site that is susceptible to breakage so that it will autocleave

UmuD’

Formed when RecA cleaves UmuD protein; the activated form called DNA pol V

Impact of mismatch repair

Increases the fidelity of DNA synthesis by 2-3 orders of magnitude

Why must mismatch repair occur quickly?

So that it is before the next round of replication to prevent permanent mutation

MutS

recognizes the mismatch

MutL

binds MutS and MutH, activates the endonuclease in MutH, recruits UrvD, and binds the beta clamp to recruit DNA pol III

MutH

an endonuclease that cuts 5’ to GATC on unmethylated strand

In E *coli*, how is the new strand identified?

Dam identifies the new strand, which distinguishes it from the old. The new strand is the one that is not yet methylates

How is the new strand identified in eukaryotes? (speculations)

There are many breaks in the lagging strand immediately after replication that could be used to identify the new strand; the leading strand has a single break in the 3’ terminus. MutS/L bind to the PNCA clamp, which could impart info on which strand is the newly synthesized one

Default repair system: MutS

has the highest affinity for the GT mismatch; repair of GT to GC

Default repair system: MutY

removes A that is paired with G=O; encodes adenosine glycosylase to create a apurinic site, which activates endonuclease and follows with an excision repair system

Default repair system: MutT

hydrolyzes 8-oxo-dGTP

Default repair system: MutM

removes G=O that is paired with C

Default repair system: 8-ox-dGTP

pairs with A

Msh2

recognizes mismatches

Msh2/3

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* binds insertion/deletion loops resulting from replication slippage (usually with a strand of A’s or T’s)

Msh2/6

binds single base mismatches, but other proteins do the repair; most prevalent

DNA slippage

Stuttering of DNA pol I where is slips backwards and synthesizes extra repeating units seen as loops sticking out of a double helix

Mechanism for recombination-repair in *coli*

Restarting a stalled replication fork using RecBC and RecA OR Repairing gaps due to thymine dimers in a daughter strand after replication by using RecR, RecO, RecA

RecBC and RecF

helps associate RecA with ssDNA

RecF/O/R

used in repairing gaps due to thymine dimers in daughter strands after replication

RecOR and RecOF

help RecA to form filaments on DNA even in the presence of SSB

How many thymidine dimers can wild-type *coli* tolerate?

up to 50

How many thymidine dimers can uvr/recA double mutant tolerate?

no more than 1-2

what triggers activated RecA?

UV irradiation

Does SOS system induce long patch or short patch repair?

long patch

is the SOS system error prone?

yes

what is the SOS system induced by?

Induced by inhibition in replication, UV damage, chemical crosslinking, and alkylating agents

LexA

Functions as a repressor; builds up and shuts down the SOS system; is activated by RecA

Lambda phage repressor

acts exactly like LexA, but instead it has an affinity for the lambda promoter

How does LexA control its own synthesis?

normally represses itself

Why is LexA repressed by LexA in terms of regulating the SOS response?

A lot of repressor LexA is needed because the SOS response is low fidelity and tolerates a lot of mistakes, which is dangerous and you don’t want that to get out of control

 promoter

A region of DNA that is upstream where proteins bind to initiate transcription

transcription unit

A sequence of DNA that codes for a RNA molecule

Polycistronic

may encode more than a single gene

How many bases are in an RNA:DNA hybrid in the bubble?

\~8-9 bp

Which strand is paired with the new RNA ?

noncoding

Why is the coding strand not the same as the template strand?

Because the template strand must be the complement of the coding strand so that the RNA produced is an exact copy of the coding strand

Rate of transcription

\~40-50 nt/sec

Rate of Translation

\~45 nt/sec

Rate of DNA synthesis

\~800 bp/sec

How does RNA pol find a promoter?

sliding, intersegment transfer and hopping models

sliding model

moves along the strand

intersegment transfer model

jumping from one strand to the next

intrasegment transfer (Hopping) model

hopping from one part of the strand to another area within the same strand

Where is RNA polymerase stored?

at nonspecific DNA sites