3-mutations and DNA repair

Biology 518 with Scot Hefty/susan Egan at University of Kansas

About this deck

By: crystal mccowen
Textbook:

Molecular Genetics of Bacteria (Snyder, Molecular Genetics of Bacteria)
Created: 2011-10-17
Size: 67 flashcards
Views: 3

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Most point mutations occur during ___ and result in ___

DNA replication

Transitions

Tautomerzation (from ___ to ___ forms) result in what type of bp change?

keto(normal) to enol

transition

(enol puring pairs with wrong pyrimidine, enol pyrimidine pairs with wrong purine)

Organisms that live at high temps will have less _ nucleotides in genome. Why?

T nucleotides

Heat promotes tautomerization of T from keto to enol form

Enol T mimics C and pairs with G

Spontaneous deamination results in what type of bp change?

transition

Deamination results in the following rxn:

C + H20 ====> U + NH2

Why do mammals often have external testicles?

Heat increases frequency of deamination (C===>U)

What enzyme recognizes U in DNA?

uracil glycosylase

Oxidation causes what type of bp change?

transversion

What altered bp is caused by oxidation? Which base does it pair with?

8-oxoguanine (from G)

pairs with A (instead of C)

5 Consequences of bp changes:

1. silent

2. missense

3. nonsense

4. loss of stop codon

5. changed regulatory element

Name the nonsense codons and their associated mutations

UAG (amber)

UAA (ochre)

UGA (opal)

Frameshift mutations occur mostly during____

repair/recombination

Frameshift mutations are caused by ____ at what type of sites?

strand slippage at series of repeated bps (either ATs or CGs)

How does a frameshift mutation affect a protein?

not leaky

all AAs past mutation are affected

usually truncated due to stop codons in new reading frame

T/F? Frameshifts revert easily

yes but usually are suppressed (not reverted) by addition/deletion of bp close to site of original mutation

How do pathogenic bacteria use frameshifts to their advantage?

take advantage of high freq and high reversion rate to change their surface antigens

escape detection by host

___% of spontaneous mutations are deletions in E.Coli

5% (considered high)

Deletions occur mostly during ____

recombination

Two common ways to produce a deletion:

1. unequal crossover = crossing over between wrong (not aligned properly) direct repeats in 2 different DNA molecules

2. looping out = crossing over between direct repeats in a single DNA molecule

Reversion rate of deletions is high/low?

very very low

Delta(lac-proAB)195 stands for ?

deletion #195 extending thru lac and proAB genes

Inversions are caused by ___

recombination between inverted repeats on the same DNA

How frequently does an inversion revert?

frequently

Effects of inversion on phenotype?

barely noticeable if at all

IN(purB-trpA)3 stands for?

inversion #3 from purB to trpA

Tandem duplication is caused by _

unequal crossing over

Frequency of tandem duplications?

frequent

Freq of tandem duplication reversions?

high freq

transposon =?

DNA elements that promote their own movement in DNA

Result of insertion on protein function?

not very leaky

almost always inactivates protein

Reversion rate of insertions?

low

Effect of deamination on the following nucleotides: C, A, G

C ===> U (pairs with A)

A ===> hypoxanthine (pairs with C)

G ===> xanthine (STILL pairs with C)

Enzymes that are responsible for repairing deaminated bases: _.

N-glycosylases

Two types of N-glycosylases?

1. Specific (for each type of deaminated base)

2. AP Lyases

Mechanism of repair by specific glycosylases?

(1) glycosylase removes deaminated base

(2) AP endonuclease makes nick in backbone on 5' side of abasic site, (3) leaving a 3'-OH that DNA POL I uses to resynthesize seq around altered base

(4) DNA Ligase seals nick

Mechanism of AP Lyases

1) AP lyase removes base and makes nick on 5' side of abasic site

2) DNA POL I uses free 3'-OH as primer to remake the seq around the altered base

3) DNA Ligase seals nick

The 3 gene products that bacteria use specifically to deal with 8-oxoguanine :

MutM

MutY

MutT

How does MutM work?

MutM is a specific glycosylase that removes 8oxo-G, cuts the strand (endonuclease activity). Strand is then degraded by exonuclease and resynthesized by DNA POL I

How does MutY work?

MutY is a specific glycosylase that removes A that's paired with 8oxo-G. Strand is then degraded by exonuclease and resynthesized by DNA POL I with C.

How does MutT work?

MutT is a phosphatase that converts free 8oxo-GTP to 8oxo-GMP so it cannot be incorporated into DNA

Two types of pyrimidine dimers?

cyclobutane ring (TT)

6-4 lesion (TC)

_____ is the repair mechanism for damage caused by UV. This mechanism doesn't occur in what types of organisms?

photoreactivation

placental mammals

Enzyme involved in photoreactivation? What type of cofactor?

photolyase (FADH2)

Mechanism of photolyase?

1. photolyase binds pyrimidine dimer in dark

2. Light activates photolyase to break dimer bonds

3. photolyase is released

______ is a general repair system that repairs only minor distortions in DNA

methyl-directed mismatch repair

Methyl-directed mismatch repair can repair what types of damage?

1. mismatches

2. base analogs

3. frameshift mutagens (planar molecules that intercalate between bps ===>addition/deletion)

4. 8oxoG

5. some types of alkylation damage

Five proteins involved in methyl-directed mismatch repair

MutS

MutL

MutH

Dam (methylase)

UvrD (helicase)

Mechanism of methyl-directed mismatch repair

1 MutS dimer binds damage in new strand

2 MutL dimer binds

3 MutH binds, uses ATP to form DNA loop, cuts new strand at GATC seq

5 UvrD separates strands, exonuclease degrades new strand past damaged base

6 DNA POL III, Ligase

8 Dam methylates new strand

How does the cell distinguish between old and new DNA strands?

parental strand is already methylated at GATC sequences. It takes a few minutes for new strand to get methylated by Dam methylase

Nucleotide excision repair: general idea?

entire damaged nucleotides are cut out and exported outside the cell

General repair system that recognizes only large distortions of DNA helix?

nucleotide excision repair

Nucleotide excision repair is used for damage caused by _______ but not by _____?

alkylating damage, any UV damage

doesn't fix: mismatches, base analogs, 8oxoG

Six proteins involved in nucleotide excision repair?

UvrA

UvrB

UvrC

UvrD

DNA POL I

DNA Ligase

DNA POL III is used for only one repair method. Which one?

methyl-directed mismatch repair

UvrABC is what type of enzyme?

endonuclease

Mechanism of nucleotide excision repair

1. UvrA2B scans DNA, UvrB binds damage

2. UvrC replaces UvrA dimer

3. UvrB cuts strand 4 nucleotides 3' from damage, UvrC cuts 7nucleotides 5' from damage

4. UvrD separates strands, removes damaged portion

5. DNA POL I remakes strand

6. DNA Ligase seals it

Describe expression of SOS genes under normal conditions

SOS genes are repressed by LexA dimer

a few SOS genes are expressed at really low levels

____ is the seq that LexA dimer binds to

sos box

How does DNA damage turn on SOS gene transcription?

1. block replication forks===> free ssDNA in cell

2. ssDNA binds RecA protein===>nucleoprotein filaments, which bind to LexA

3. autocleavage of LexA===>dissociates from SOS box

4. transcription of sos genes

What is special about DNA POL V?

can replicate right over damaged spots, just inserts random bases

(capable of translesion synthesis (TLS))

UmuC and UmuD are examples of ____ genes that form what enzyme?

SOS genes

DNA Pol V (inactive if UmuD2C, active if UmuD'2C)

What happens after LexA has autocleaved and UmuC/D have been translated?

1. UmuD2C forms (cannot polymerize but can arrest replication by DNA Pol III)

2. Once [RecA nucleoprotein filaments] is high enough, UmuD autocleaves itself ===> UmuD'2C (active DNA Pol V)

Why does it take a higher [RecA nucleoprotein filaments] to cause UmuD to autocleave than LexA?

DNA Pol V is only activated if the damage is so bad that it cant be immediately repaired by other SOS functions

Mechanism of UmuD'2C Translesion Synthesis:

1 Pol III stalls at damage, helicase keeps going

2 RecA polymerizes on ssDNA==> RecA nucleoprotein filmant coat

3 UmuD'2C is recruited, clamp transferred from Pol III to V. Pol III is displaced.

4 Pol V replicates over damage,~5bps, Pol III resumes

Functions of the RecA "coat" on the ssDNA?

1. attracts UmuD'2C to site

2. Pol V is more efficient in presence of RecA

3. Pol V is less error-prone in presence of RecA

How do DNA Pol III and V compare on non-damaged DNA?

Pol V is more error prone on non-damaged DNA ===>untargeted mutations

What is ironic about umuC/umuD mutants?

they are just as well off as wild-types when exposed to mutations

About this deck

By: crystal mccowen
Textbook:

Molecular Genetics of Bacteria (Snyder, Molecular Genetics of Bacteria)
Created: 2011-10-17
Size: 67 flashcards
Views: 3

About StudyBlue

“Simply amazing. The flash cards are smooth, there are many different types of studying tools, and there is a great search engine. I praise you on the awesomeness.”
Dennis