DNA replication

Question 1

Overview of steps:

Answer 1

Recognition of replication origin by origin binding proteins
Helicase melts/unwinds DNA
Topoisomerase/gyrase relaxes torsional stress ahead of the replication fork
SSB bind to protect unwound ssDNA
RNA primase lays down RNA primers
DNA Pol III elongates / lays down nts
DNA Pol I removes RNA primers & lays down DNA
DNA Ligase ligates the fragments together

Question 2

What exonuclease activity do Pol I & III have?

Answer 2

Both Pol I & III have 3’ –> 5’ exonuclease activity, so they can “back up” and fix a mistake. Only Pol I has 5’ –> 3’ exo. activity so it can remove those RNA primers as it goes.

Question 3

How does DNA Pol make a phosphodiester bond?

Answer 3

It breaks off a diphosphate group from the dNTP and uses that energy to link the remaining phosphate to the hydroxyl group.

Question 4

Where is the sliding clamp of Pol III located in prokaryotes and eukaryotes.

Answer 4

Eukaryotes: PCNA of Pol delta
Prokaryotes: beta subunit of Pol III

Question 5

In eukaryotes which polymerases are responsible for the leading & lagging strands?

Answer 5

Pol Pol delta does leading strand; epsilon does lagging strand.

Question 6

What do ORI sequences look like?

Answer 6

Typically AT-rich regions so they can be pulled apart easily.