Dna Replication

Front 1

Describe the OriC in E. Coli. specifically
a)number of bp.
b)describe the consensus sequences (2) in OriC (location, # of bp, # of repeats, and function.

Back 1

OriC-E. Coli chromosomal replication origin
A)consists of 245 bp
B) 1)3 repeats of 13 bp A/T rich region on left edge; act as sites for opening dsDNA for initiation and loading of DnaB helicase. 2)4 repeats of DnaA box, 9bp sequences that act as recognition sites for DnaA binding

Front 2

What is the role of a)DnaA, b)DnaG, c)DnaB, d)HU protein, e)DnaC, f)RNA polymerase, g)SSB protein, h)Dam methylase, i)DNA gyrase in E. Coli replication initiation?

Back 2

a)DnaA-recognizes ori sequence and opens (melts) double stranded DNA at origin
b)DnaG-Synthesizes RNA primers (primase)
c)DnaB-Unwinds dsDNA
d)HU-Histonelike protein, stimulates initiation and negative loop formation w/ DnaA
e)DnaC-loads DnaB onto the DNA
f)RNA polymerase-activates oriC, necessary for DnaA access to DNA
g)SSB protein-binds and protects ssDNA
h)Dam methylase-Methylates (5')GATC sequences at oriC
i)DNA gyrase-(prokaryotic topoisomerase II) relieves torsional strain from DNA unwinding.

Front 3

What are the proteins of the primosome complex

Back 3

n,n',n'',DnaB,DnaC,DnaT,DnaG

Front 4

The DNAP III holoenzyme contains several subunits. What are these enzymes and what is their role?

Back 4

DNA Pol III holoenzyme consists of the alpha, epsilon, beta, tau, gamma, delta, theta, chi, and psi.
alpha-possesses 5'-3' polymeryzation activity.
epsilon-3'-5' proofreading exonuclease activity.
beta-clamp subunit, enhances processivity
tau-mediates coupling of dimeric DNAP III complex (keeps leading and lagging strand duplex tethered)
gamma-clamp loading complex
other subunits not specified

Front 5

How does ciprofoxacin affect DNA replication?

Back 5

Ciprofloxacin binds to DNA gyrase, inhibiting its ability to releive torsional strain on the DNA molecule during replication and subsequently inhibiting cell division.

Front 6

What is the length of Okazaki fragments in prokaryotes? eukaryotes?

Back 6

1000-2000 nt in prokaryotes
100-200 nt in eukaryotes

Front 7

What is the role of
a) DNAP III
b) DNAP I
c) DNA ligase

Back 7

a)DNAP III polymerizes leading and lagging strand (highly processive)
b)DNAP I-removes RNA primers w/5'-3' exonuclease activity and replaces w/ DNA w 5'-3' polymerase activity
c)DNA ligase seals the nick that remains after DNAP I has replaced RNA primers w/ DNA

Front 8

Briefly describe the process of DNA replication termination in prokaryotes?

Back 8

Seven ter sites (~20bp) located on the opposite side of the chromosome from oriC bind Tus proteins (termination utilization substance) which stops the continuation of replication forks. The 2 daughter strands are then decatenated by topoisomerase 4 (a type II isomerase)

Front 9

What are the 4 biochemical requirements for DNA polymerase activity?

Back 9

a)polynucleotide template
b)primer w/ a free 3"-OH annealed to template
c)dNTPs readily available
d)polymerization reaction (dNMP)n + dNTP=(dNMP)n+1 + PPi (pyrophosphate is subsequently hydrolyzed)

Front 10

What is the
a)average synthesis rate
b)average processivity
of DNAP I? and (c,d) of DNAP III?
e) What is the third biochemical activity of DNAP I that DNAP III lacks?

Back 10

a)20nt/sec (16-20)
b)50nt (3-200)
c)500-1000nt/sec
d)>500,000
e)5'-3' exonuclease activity (for primer removal)

Front 11

a)What cofactor does E. Coli DNA ligase use?
b)What cofactor do viral and eukaryotic DNA ligase use?
c)Which DNA ligase can ligate blunt end DNA?
d)What is the mechanism of DNA Ligase action?

Back 11

a)NAD+
b)ATP
c)Bacteriophage T4 DNA Ligase
d) i)DNA ligase is adenylated by cofactor, ii)activation of 5'phosphate of nick by addition of adenylate (AMP), iii)nucleophilic attack by 3'OH of nick releases AMP and seals the nick.

Front 12

There are multiple origins of replication in Eukaryotic chromosomes. How far apart are they usually?

Back 12

30,000 to 300,000 bp

Front 13

What are the roles of the different eukaryotic DNA polymerases?

Back 13

DNAP alpha-primase activity-synthesizes RNA primers, also 5'-3' polymerase activity
DNAP beta-DNA repair
DNAP delta-elongates leading/lagging strand
DNAP gamma-mitochondrial DNA replication
DNAP epsilon-candidate for leading/lagging strand synthesis, also DNA repair and primer removal

Front 14

There are several eukaryotic proteins that are analogous to the proteins involved in prokaryotic replication. What are the analogous proteins for a)DNAP III beta subunit, b)DnaC, c)DnaB, d)SSB, e)DNAP III gamma subunit

Back 14

a)DNAP III beta=PCNA (Proliferating Cell Nuclear Antigen)
b)DnaC=Cdt1/CdC6
c)DnaB=Mcm2-7
d)SSB=RPA
e)DNAP III gamma=RFC

Front 15

What are the roles of the following proteins in eukaryotic DNA replication?
a)ORC, b)Cdc6/Cdt1, c)Mcm2-7, d)CDK, e)Crm1, f)Geminin, g)Mcm10, h)DDK/CDK, i)Cdc45/Sld3,

Back 15

a)ORC=6 subunit complex that binds that initiates replication
b)Cdc6/Cdt1-recruits and loads the Mcm2-7 helicase complex
c)Mcm2-7=DNA helicase
d)CDK-phosphorylates Cdc 6
e)Crm1-exports the phosphorylated Cdc6 outside the nucleus
f)Geminin-removes Cdt1 from DNA and initiation complex
g)Mcm10-recruits DDK/CDK to complex
h)DDK/CDK-recruit Cdc45/Sld3
i)Cdc45/Sld3-bind to complex followed by RPA,Primase, and finally pol delta. DDK/CDK are removed from DNA after RPA binding.

Front 16

a)What is the role of Telomerase?
b)What is its mechanism of action?

Back 16

a)Telomerase is responsible for elongating reiterating regions located at the ends of linear chromosomes
b) i)Telomerase extends the 3'end of DNA using the RNA of telomerase as a primer for reverse transcription (RNA->DNA) (several rounds)
ii)Primase adds RNA primer to the newly extended 3'end
iii) the newly extended 3'end serves as a template for DNA polymerase
iv)RNA primer is removed

Front 17

In eukaryotes, how are telomeres protected from nucleases and repair enzymes?

Back 17

The telomere is folded into a T loop with the single stranded portion invaded the dsDNA with which it is complementary (remember telomeres are repeat sequences). TRF1/2 bind the loop and stabilize it.