19/ dna replication

Question 1

direction of dna synthesis

Answer 1

5’-3’
antiparallel to template

Question 2

key components of e coli/human replication form (conserved) DIAGRAM

Answer 2

• on leading strand, sliding clamp w dna polymerase (heading into parental dna)
• on lagging strand, single strand dna binding protein, sliding clamp and clamp loader
• At fork, dna helicase, and dna primase (where next Okazaki fragment will extend from) - together primosome
• other enzymes: ribonuclease H, dna ligase, dna topoisomerases I and II

Question 3

briefly how are the leading and lagging strands synthasised

Answer 3

• leading - continuous 5’ to 3’
• lagging - discontinuous 5’-3’

Question 4

synthesis of primers for dna replication

Answer 4

• short rna primer synthesised using a template and nucleotide triphosphates by dna primase
• once this is in place, dna polymerase extends it
• requires primer template junction

Question 5

differences in lagging strand synthesis

Answer 5

• dna primase makes rna primer as normal, and dna polymerase extends
• ribonuclease H removes primer (leaves a gap in the strand)
• dna polymerase extends across gap
• dna ligase seals nick

Question 6

what does dna helicase do? is it active or passive?

Answer 6

• sperates parental dna strands at the replication fork and move replication fork forwards
• active - uses atp

Question 7

werner syndrome.

Answer 7

• progeria (premature aging)
• autosomal recessive
• mutation in RECQ helicase gene WRN
• we dont get seperation of replication fork
• insufficient replication. cells senesce rather than replicate - hallmark of aging

Question 8

bloom syndrome

Answer 8

• cancer syndrome - predisposition to cancer
• LoF mutation in RecQ family DNA helicase which maintains genome integrity
• redness on face

Question 9

what is the processivity of an enzyme

Answer 9

how fast an enzyme can work. no of consecutive reactions w/o letting go of substrate

Question 10

how is processivity of dna polymerase increased? what does this mean

Answer 10

• association with sliding clamp
• once 1st step of dna synthesis has been done, interaction of enzyme w primer template junction is maintained and addition of further nucleotides is rapid - can add thousands rather than 1
• sliding clamp keeps polymerase bound to dna
• atp dep
• positioned close to primer template junction by clamp loader

Question 11

structure of e coli sliding clamp, what is this very similar to

Answer 11

• encircles dna like a nut on a bolt or a donut
• human sliding clamp: proliferating cell nuclear antigen pcna

Question 12

role of single stranded dna binding proteins SSBs

Answer 12

• expose single stranded dna in the replication fork, so it can be used as a template and easing replication fork progression
• enhance processivity of dna polymerase by keeping fork open

Question 13

how do SSBs monomers bind to dna? what does this helpfully prevent

Answer 13

• dna base pairs can bind to each other within the same strand, creating hairpins
• ssbs coat the single strand of dna, preventing this
• they bind cooperatively

Question 14

role of dna topoisomerases

Answer 14

• prevent dna from becoming tangled during replication, thus enhancing processivity of dna polymerase
• when helicase unwinds, creates a superhelical tension in the dna - supercoiling. this needs to be relaxed

Question 15

mechanism of type 1 and 2 dna topoisomerases

Answer 15

• nick and reseal backbone of parental helix
• type 1: nick and reseal 1 dna strand, no atp needed
• type 2: nick and reseal both strands, atp needed

Question 16

compare origins of replication in yeast and humans

Answer 16

• yeast: autonomously replicating sequences ARS
• huamns: poorly understood, sequences near to LMNB2, MYC and also defined by chromatin structure (no histones)

Question 17

how many origins of replication in e coli, yeast, humans

Answer 17

• e coli - 1
• yeast - 600-700
• humans - over 100,000

Question 18

biphasic initiation of dna replication in eukaryotes, what ensures each origin is used and each chromosome is only replicated once per cycle

Answer 18

• replicator selection occurs in g1 phase (formation of pre-Replicative Complex). but nothing is activated yet
• origin activation in S phase - unwinding of DNA and recruitment of dna polymerase
• temporal separation

Question 19

more detail of process of G1 eukaryotic replicator selection, resulting in pre-replicative complex

Answer 19

• origin recognition complex (ORC) binds to replicator sequence (unknown in humans, ARS in yeast)
• helicase loading proteins Cdc6 and Cdt1 bind to ORC
• this activates helicase Mcm2-7 to bind and complete formation of pre-RC
• INACTIVE - just preparing here

Question 20

what does cyclin dep kinase Cdk activity in s phase do to pre-RCs

Answer 20

• activates existing, does NOT form new pre-RCs
• levels are low in g1 (preventing activation) but high in s (preventing formation)

Question 21

what is the problem (2) when you get to the chromosome ends for finishing dna rep, and the solution

Answer 21

• dna polymerase and ligase close all but one gap
• nothing upstream so no polymerase to synthasise over, one overhang on start of leading and end of lagging
• ribonuclease H removes rna primers - further shortening newly synthasised dna strands at 5’ ends of chromosomes
• telomeres! add TTAGGG repeats
• extended 3’ end dna now long enough to enable dna primase to bind and initiate new rna primer synthesis, can be extended as extra okazaki frag by dna polymerase

Question 22

how does telomerase know to add its repeat/how does it work

Answer 22

• ribonucleoprotein that contains rna component that specifies telomere sequence - acts as template
• repeat sequences sythasised in a step-wise process
• telomerase shuffle - moves along and rebinds at newly synthasised ttagg