Lecture 6: DNA Metabolism Flashcards
ispA
Gene or Protein
Gene
IspA
Gene or Protein
Protein
Meselson-Stahl demonstrated what in regards to the structure of DNA
Semi-conservative; there are two strands of DNA
______(Enzyme) ___ are responsible for replication
DNA Polymerase
DNA Template runs in what direction?
3” -> 5’
DNA Primer run-in what direction?
5’ -> 3’
DNA Polymerase runs in what direction?
5’ -> 3’
How does DNA Polymerase bind to the stand? What mechanism occurs?
DNA Polymerase attacks alpha-phosphorous group
Phosphate group comes off
Forms a phosphodiester bond
DNA Polymerase utilizes single stranded DNA templates and adds nucleotides to a growing chain in the _____ direction.
5’ -> 3’ direction
What is the ORI (Origin of Replication?
Unique point where DNA replication begins
What type of N-Bases are found at the ORI?
A-T rich
E. Coli replicates using what mechanism?
Replication Bubbles
Replication forks travel in what direction?
Bidirectional
The branch point in a replication bubble is called _______.
Replication Fork
New DNA is added to which end of the strand?
3’ end
Leading Strand reads?
5’ -> 3’
Lagging Strand reads?
3’ -> 5’
What is DNA Ligase?
Enzyme that seals/joins the discontinuous Okazaki fragments together into a continuous piece of DNA
Okazaki Fragments are found on which strand?
Lagging
What is Primase?
Synthesize RNA Primers
What is semi-conservative replication?
Each strand includes one parent strand and one newly synthesized daughter strand
What is exonuclease?
Remove DNA only from the ends of DNA strands
What is endonuclease?
Degrade DNA from the interior of a DNA strand by hydrolyzing the phosphodiester bond)
What are restriction enzymes?
The specified sequences where endonuclease cut internally
What is DNA Polymerase?
Enzyme that synthesize DNA
True or False
DNA Polymerase will polymerase 5’ -> 3’
True
DNA Polymerase is an favorable or unfavorable?
Favorable
exergonic
(-) Delta G
Does DNA Polymerase require a primer?
Yes
E. coli DNA polymerase III:
A) can initiate replication without a primer.
B) is efficient at nick translation.
C) is the principal DNA polymerase in chromosomal DNA replication.
D) represents over 90% of the DNA polymerase activity in E. coli cells.
E) requires a free 5’-hydroxyl group as a primer
C
ORI is A-T rich or G-C rich?
A-T
. The Meselson-Stahl experiment established that:
A) DNA polymerase has a crucial role in DNA synthesis.
B) DNA synthesis in E. coli proceeds by a conservative mechanism.
C) DNA synthesis in E. coli proceeds by a semiconservative mechanism.
D) DNA synthesis requires dATP, dCTP, dGTP, and dTTP.
E) newly synthesized DNA in E. coli has a different base composition than the preexisting DNA.
C
When a DNA molecule is described as replicating bidirectionally, that means that it has two:
A) chains. B) independently replicating segment. C) origins. D) replication forks. E) termination points.
d
An Okazaki fragment is a:
A) fragment of DNA resulting from endonuclease action.
B) fragment of RNA that is a subunit of the 30S ribosome.
C) piece of DNA that is synthesized in the 3’—>5’ direction.
D) segment of DNA that is an intermediate in the synthesis of the lagging strand.
E) segment of mRNA synthesized by RNA polymerase.
d
The proofreading function of DNA polymerase involves all of the following except:
A) a 3'--->5' exonuclease. B) base pairing. C) detection of mismatched base pairs. D) phosphodiester bond hydrolysis. E) reversal of the polymerization reaction.
E
The 5’—>3’ exonuclease activity of E. coli DNA polymerase I is involved in:
A) formation of a nick at the DNA replication origin.
B) formation of Okazaki fragments.
C) proofreading of the replication process.
D) removal of RNA primers by nick translation.
E) sealing of nicks by ligase action.
D
Prokaryotic DNA polymerase III:
A) contains a 5’—>3’ proofreading activity to improve the fidelity of replication.
B) does not require a primer molecule to initiate replication.
C) has a beta subunit that acts as a circular clamp to improve the processivity of DNA synthesis.
D) synthesizes DNA in the 3’—>5’ direction.
E) synthesizes only the leading strand; DNA polymerase I synthesizes the lagging strand.
C
Which of the following is not required for elongation during DNA replication in E. coli?
A) DnaB (helicase) B) DnaG (primase) C) DnaC D) Beta-sliding clamp E) Clamp loader
C
The function of the eukaryotic DNA replication factor PCNA (proliferating cell nuclear antigen) is similar to that of the -subunit of bacterial DNA polymerase III in that it:
A) facilitates replication of telomeres.
B) forms a circular sliding clamp to increase the processivity of replication.
C) has a 3’—>5’ proofreading activity.
D) increases the speed but not the processivity of the replication complex.
E) participates in DNA repair.
B
. In a mammalian cell, DNA repair systems:
A) are extraordinarily efficient energetically.
B) are generally absent, except in egg and sperm cells.
C) can repair deletions, but not mismatches.
D) can repair most types of lesions except those caused by UV light.
E) normally repair more than 99% of the DNA lesions that occur.
E
Which of these enzymes is not directly involved in methyl-directed mismatch repair in E. coli?
A) DNA glycosylase B) DNA helicase II C) DNA ligase D) DNA polymerase III E) Exonuclease I
A
. Which mechanism is used to repair a thymidine dimer in DNA?
A) Mismatch repair B) Base-excision repair C) Nucleotide-excision repair D) Direct repair E) More than one is used for this type of lesion
D
Which mechanism is used to repair a chemically modified base in DNA?
A) Mismatch repair B) Base-excision repair C) Nucleotide-excision repair D) Direct repair E) More than one is used for this type of lesion
E
In homologous recombination in E. coli, the protein that assembles into long, helical filaments that coat a region of DNA is:
A) DNA methylase. B) DNA polymerase. C) histone. D) RecA protein. E) RecBCD enzyme.
D
Which of the following is not a feature of homologous recombination during meiosis?
A) A double strand break
B) Cleavage of two crossover events
C) Alignment of homologous chromosomes
D) Formation of a single Holliday intermediate
E) Exposed 3’ ends invade the intact duplex DNA of the homolog
D
True or False;
Most polymerases have 3’ -> 5’ exonuclease activity, which is used to remove incorrect bases; proofreading capability
True
In E. Coli, DNA Polymerase III is ___
Replication Enzyme.
In E. Coli, DNA Polymerase I is ____.
clean up functions during replication, recombination and repair.
In E. Coli DNA Polymerase II, IV, and V ___.
DNA Repair
In E. Coli, which DNA Polymerase is the fastest?
Polymerase III
In E. Coli, Which DNA Polymerase participates in nick translation?
DNA Polymerase I
What is nick translation?
Removes a short stretch of RNA or DNA and replaces it with a new sequence of DNA
Why can DNA Pol I have the ability to be involved in nick translation?
It contains a subunit which contains a 5’ -> 3’
What are the three parts of DNA Poly III?
Core Polymerase
Clamp-Loading Complex
(CLAW)
Clamp
In E.Coli, DNA Primase does what?
Creates RNA primers on Okazaki Fragments in E. Coli
In E. Coli, does DNA replication involve an RNA Primer?
Yes
What are SSB?
Single -Stranded Binding Proteins; keeps DNA from rewinding.
What are DNAB?
Helicase; unwinds DNA
DNA Gyrase
Negative supercoils into DNA as it is being replicated, allowing process to proceed.
What are the three conformations of DnaB?
- NTP Bound
- hydrolysis
- release of products?
True or False;
DnaB travels on both strands simultaneously
False; DnaB (Helicase) only travels on a single strand at a time in the 5’ -> 3’ direction
True or False;
SSB are monomeric proteins that coats ssDNA (single-stranded DNA)
False; SSB are tetrameric
What is DNA Ligase?
Ligates two pieces of DNA together to form a contiguous stand.
An adenylate moiety is added to the ______ to promote ligation.
5’- phosphoryl terminus
True or False
DNA Ligase is an example of Adenylalation?
True
Adenylalation of Substrate and Enzyme happens in what process?
DNA Ligase in nick sealing
E. Coli replication can be divided into what steps?
Initiation
Elongation
Termination
E. Coli Initiation takes place at _____.
Origin of Replication
DnaA Protein
Recognizes oriole’s sequence, opens duplex at specific sites in origin
DnaB protein (helicase)
Unwinds DNA
DnaC protein
Required for DnaB binding at origin
HU, FIS, IHF
Histone-like protein; DNA-binding protein, stimulates initiation
DnaG (Primase)
Synthesizes RNA Primers
SSB (Single Stranded Protein)
Binds single-stranded DNA
DHA Gyrase
Relieves torsional strain generated by DNA unwinding
DNA methlase
Methylates (3’) GATC Sequences at otic
Consensus Sequence
What is most definitely found at a certain sequence when compared with others.
What are the two major steps in E. Coli Replication Initiation
- Binding of an ATP-bound Dana to five-9 bp repeats in the otic
- This complex then denatures the DNA in 3-13 bp DUE sequences also floated in the oriC
True or False;
ATP dnaA is active during initial E. Coli Replication Initiation
True
How does dana recognize consensus sites?
Base-specific interactions in major grooves; alpha 5’-helix sits in the major groove.
What are the importance of the Minor Groove?
Assists protein in binding
Interaction with backbone phosphate in major groove _____.
Enhancing interactions
ADP dnaA is _____>
not active
ADP &ATP bound dnaA can bind ____ in ____.
R-Sites
oriC
ATP-Bound dnaA can bind ___.
I-sites
HU, FIS & IHF prevents unwinding of DNA
False; they all assist/help with unwinding
Sequence of Events:
E. Coli Replication Initiation
- dnaC loads dnaB into unwound segment of DNA
2. dnaB (Helicase) unwind DNA in both directions, creating replication forks.
Dam Methylase
Enzyme that methylates oriC DNA of N6 of adenine residues in the GATC sequences.
What is hemi-methylated
Only one of the strands in methylated; prevents replication from beginning again.
Methylation is regulating___-
When DNA replication can occur again.
What is DnaG
Lays down short RNA primers at the origin; aka Primase
What is DNA Pol III
Begins adding deoxyribonucleotides in a continuous fashion to create the leading strand.
True or False
DNA Polymerase interacts wth DnaC and travels along the replication fork.
DnaB
What is synonymous with dnaB
Helicase
Binding site of SSB is lined with ______.
Hydrophobic Aromatic Residues
In E. Coli DNA Poly. III, is it a monomer or dimer?
Dimer
What is another name for dnaG?
Primase
When ______ is completed, _____ removes the RNA Primer and replaces it with _____.
Okazaki Fragments
DNA Pol I
DNA
_____ seals the nick to make a continuous piece of DNA.
DNA ligase
How does the Beta Clamp know how to associate with RNA-DNA hybrid?
Hydrophobic Pocket in proteins
What is the fx of Topoisomerase IV?
separates the two chromosomes and they are segregated into daughter cells
True or False;
Eukaryotic Replication involves circular replication.
False; linear replication
True or False;
Eukaryotic Replication is faster than Prokaryotic.
False; it is slower.
__________-
Permanent changes in the genomic nucleotide sequence and can be due to base changes or the addition or deletion of base pairs.
Mutations
What is the Ames test?
measures rate of mutations
What is the difference between carcinogen and mutagen?
Carcinogen mutates your genome in a position that causes cancer
Mutagen mutates your genome
What are the 4 repair systems
Mismatch
Base-Excision
Nucleotide Excision
Direct
______ corrects DNA using the sequence of the template strand, so utmost be able to determine “old DNA” from “new DNA’
Mismatch Repair
How is Mismatch Repair able to differentiate between old and new DNA?
Methylation of adenine residues;
Original strand is methylated; usually takes a few minutes after replication to methylate the new strand.
Mut Repair System is used in which DNA repair system?
Mismatch Repair
Which two proteins form a complex and bind to the mismatch site?
MutL & MutS proteins
Which protein in Mismatch Repair recognizes and determines mismatch and which residue does it work with?
MutS;
Phenylalanine36 residue pushes against base pairs.
if weak, mismatched, bases will swing into the syn configuration. MuS recognizes mismatched base pairs by swinging them from anti-syn configuration
In Mut Repair System, which protein has endonuclease activity?
MutH
Which Mutation Repair System Protein system shred DNA through complex?
MutL
MutH
MutS
What is the Fx. of DNA Glyoxylase in Base-Excision Pair?
removes modified base by cleaving N-glycosyl bond to make an apurinic/apyrimidinic site (AP site)
What is the Fx. of AP endonuclease in Base-Excision Pair?
removes deoxyribose 5’ phosphate
What are the 3 steps in Base-Excision Repair
- DNA Glycosylase removes damaged base by cleaving N-glycosyl bond at AP site.
- AP Endonuclease removes deoxyribose 5’ phosphate left behind by cleaving phosphodiester bonds.
- DNA Polymerase I replaces the removed DNA before DNA ligase seals the remaining nick.
What is the Nucleotide Excision Repair
Repair large distortions in the helical structure of DNA.
What is the ABC Exinuclease and what repair system is it assoc. with?
Hydrolyzes 2 phosphodiester bonds, one on either side of the distortion; Nucleotide Excision Repair
What enzymes are assoc. with ABC Excinuclease?
Uvr proteins A, B, C and D
Which repair system would you use during methylation?
Direct Repair
Which repair system Utilizes/ Sacrifices enzyme to rid of methyl group?
Direct Repair
What is SOS repair system?
Excessive DNA damages activates SOS response in bacteria
True or False;
DNA Polymerase V can lay down nucleotides without a template.
True
Which DNA repair system utilizes DNA photolyases?
Direct Repair
Xeroderma is linked to people who are _____ and are linked to which repair system?
Light sensitive; Nucleotide Excision Repair
