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 III
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)
NO PRIMER REMOVAL - 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?
- monomeric
- is E.coli DNA Pol III monomeric or heteromeric?
- 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?
-
DnaA
DnaB (helicase)
DnaC
DnaG
SSB - 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?
-
NO
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?
-
DNA Pol III
topoisomerases
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
DMS
grilling - what happens when you deaminate cytosine?
- uracil
- what happens when you deaminate adenine?
- hypoxanthine
- what happens when you deaminate guanine?
- xanthine
- what is base excision repair?
-
incorrect/damaged base is recognized and removed (can occur OUTSIDE of S phase)
polymerase replaces base
ligated - 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?
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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
also,
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