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Biochem - DNA Synthesis and Repair


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what do helicases do?
unwind DNA
what do topoisomerases do?
remove supercoils
what does processivity mean?
staying on the strand and going a long distance
what kind of bonds are formed in DNA synthesis?
ester bond b/w 5'phosphate of incoming NTP and free 3'OH on growing chain (3'->5' bond)

pyrophosphate released
what drives DNA synthesis?
hydrolysis of pyrophosphate by pyrophosphatases (PPi -> 2Pi) drives DNA synthesis forward
what DNA polymerase does E.coli use?
Pol I

Pol II, Pol IV, and Pol V as well for DNA repair
what is E.coli DNA Pol I used for?
repair and replication

5'-3' elongation
3'-5' exonuclease (proofreading)
5'-3' exonuclease (primer removal)
what is E.coli DNA Pol III used for?
Pol III is used for replication

5'-3' elongation
3'-5' exonuclease (proofreading)

how is the processivity of E.coli DNA Pol I?
slow, low processivity
how is the processivity of E.coli DNA Pol III?
fast, high replication rate and processivity
what are E.coli DNA Pol II, IV, and V used for?
DNA repair
is E.coli DNA Pol I monomeric or heteromeric?
is E.coli DNA Pol III monomeric or heteromeric?
where does the replication fork initially generated for prok?
oriC genetic locus
what does the primosome do?
initiates DNA synthesis in PROKARYOTES

unwinding, stabilizing ssDNA, and initial synthesis of RNA primers
what are the components of the primosome?
DnaB (helicase)
what does DnaA of the primosome do?
tetrameric DNA binding protein with specificity for oriC

requires ATP

directs other primosome proteins to origin

initial melting of short AT rich region
what does DnaC of the primosome do?
required for DnaB (helicase) binding
what does DnaB do?
it is a heolicase

separates duplex DNA

ATP gets hydrolyzed
what does DnaG do?
it is the primase

required to generate RNA primers
when does the DnaA tetramer get displaced?
after helicase (DnaB), SSB, and primase (DnaG) bind to DNA
can prokaryotic DNA polyermases initiate DNA synthesis?

needs DnaG in primosome to create RNA primers
what does the replisome do?
Elongation during prokaryotic DNA synthesis
what are the components of the replisome?

with help from:
DNA Pol I and DNA Ligase
what does DNA Pol III do?
it is recruited to the primosome and REQUIRED for elongation

elongates RNA primer; processive
what do fluoroquinolones do?
they are bacteriacidal drugs that inhibit gyrase (a topoisomerase) in prokaryotes

i.e. cypro
who does the proofreading (3'-5' exonuclease) in prokaryotes?
Pol I and Pol III
when does termination of DNA synth in prok occur?
when replication forks meet at TER SEQUENCE repeats

proteins bind and arrest forks by inhibition of DnaB
what are the polyermases used in DNA synthesis of eukaryotes?
alpha (initiation)
beta (initiation)
delta (elongation)
epsilon (repair)
gamma (mit replication)
which of the DNA polyermases in euk have 3'-5' exonuclease activity?
delta, epsilon, and gamma
who does the eukaryotic primer removal (5'-3' exonuc)?
RNase H and Fen 1

NOT by a polyermase (diff from prok!)
what makes euk DNA synthesis more complicated than in prok?
larger size
complicated organization
thousands of origins of replication
what are telomeres?
non-encoding sequences at end of linear chromosomes

hundreds of tandem repeats (TTAGGG in vertebrates)
what is the purpose of telomeres?
maintain structural integrity of euk chromosomes

w/ each round of DNA replication, shorter daughter DNA
what is telomerase?
a template bearing reverse transcriptase that extends telomeres

contains RNA template (matches G rich telomeres) and a protein w/ polymerase activity
which cells lack telomerase?
human somatic cells

(germ and early embryonic are constantly dividing)
what is Hutchinson-Guilford Progeria?
premature aging w/ short telomeres
why do you need DNA repair?
DNA Pol "proofreading" is not perfect

spontaneous damage (i.e. depurination)

agents (UV, oxygen radicals, alkylating agents that methylate, depurination, deamination, intercalating agents)
what are some alkylating agents that can methylate DNA?
dimethyl sulfate
what happens when you deaminate cytosine?
what happens when you deaminate adenine?
what happens when you deaminate guanine?
what is base excision repair?
incorrect/damaged base is recognized and removed (can occur OUTSIDE of S phase)

polymerase replaces base

describe the E.coli methyl-directed mismatch repair system of Base Nucleotide Excision Repair in prokaryotes
1. parent strand w/ GATC seq is recognized

2. methyl grp added to A by DAM (DNA adenine methylation) methylase

3. Mut proteins recognize and repair mismatch of UNDER-METHYLATED (new) strand
What are the Mut proteins used in E.coli methyl-directed mismatch repair?
MutS - recognizes and binds damaged DNA

MutL - joins and promotes loop structure; also endonuclease

MutH - recruited to recognize GATC (methylated) sites and strand specificity; endonuclease and exonuc removes NMPs
which strand is hypermethylated, the parent or newly-synthesized?
the parent
describe the E.coli UVR system in bacterial nucleotide excision repair
1. recognizes pyrimidine dimers and other bulky lesions (drugs)

2. uses EXInuclease (both endo and exo) - dual cut that requires ATP
what are the subunits of the UVR endonuclease protein?
UvrA, UvrB, UvrC

w/ help from UvrD, DNA Pol, and DNA ligase
what does UvrA do?
Damage sensing
UvrB loading
helicase subunit

Functional motifs:
ATP binding, zinc fingers, helix-turn-helix
what does UvrB do?
helicase subunit
binds damage site after UvrA
nuclease subunit

Functional motifs:
ATP binding, 3' nuclease
what does UvrC do?
damage specific nuclease

functional motif:
5' nuclease
what are the steps of the E.coli UVR system in bacterial nucleotide excision repair?
1. UvrA recognizes damage, then recruits UvrB

2. Helicase of UvrAB unwinds DNA;

3. UvrA dissociates and UvrC associates, activating exinuclease

4. UvrD displaces fragment; DNA Pol and DNA ligase resynthesizes damaged strand
what is an example of bacterial base excision repair?
E.coli DNA glycosylase-requiring repair system
what are the steps in E.coli DNA glycosylase-requiring repair system?
1. damaged base is cleaved from deoxyribose by a DNA glycosylase enzyme

2. apurinic/apyrimidinic (AP) deoxyribose is released by AP endonuclease and AP exonuclease (removes sugar-phosphate)

3. gap filled and nick sealed by ligase

there are a NUMBER of enzymes, each w/ SPECIFICITY for specific damaged base
how is mammalian excision repair similar to prok?
euk cells have functional and structural homolgues to Mut proteins

hMLH and hMSH

however, no MutH homolog found yet

functionally analogous base excision repair enz.
how is mammalian excision repair different from prok?
structurally different exinucleases (functionally similar)

human exinuclease cleave LARGER # bases surrounding lesion
what is Xeroderma Pigmentosum?
UV induced thymine dimer repair

defect in any of 7 proteins (XPA-XPG)
what does ionizing radiation cause?
double stranded break

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