DNA Replication Part II

Week 4

Initiator Proteins

Require ATP to wrap themselves in and melt some H-bonds, which attracts Helicase protein. They also help helicase bind.

How many helicase proteins are there? Which one is predominant?

Two helicase proteins, predominant helicase moves in the 5’ → 3’ direction along lagging strand template.

What does helicase require to function?

ATP, in order to break into ADP + Pi when separating base pairs

What level of organization is the helicase protein?

Quaternary

Single-strand binding proteins

Are monomers. Prevents re-annealing double-stranded templates (forming H-bonds) without blocking DNA Polymerase

* Partial H-bonds can cause hairpin loops

What are RNA primers

* Short sequences of nucleotides with a free 3’ OH base-paired to template - required by DNA polymerase to start replication

Purpose of primase in DNA replication

* Synthesize an RNA primer with free 3’ OH that DNA pol. can use. i.e., 5’ → 3’

Primase proceeds in which direction?

* reads 3’ → 5’ along template strand, same as DNA pol.

5 Steps of bacterial DNA Replication

1. Origin of replication.
2. Binding of initiator proteins.
3. Unwinding by helicase.
4. Binding of single-strand binding protein.
5. RNA primers made by primase.

Sliding clamp function

* Holds onto DNA pol. from behind to prevent it from falling off.
* Ring shaped

@@Describe and label a replication fork@@

How are Okazaki fragments on the lagging strand linked together?

* A special DNA repair system is responsible for removal of the RNA primer and replacing it with a correctly matched DNA sequence
* repair pol. uses og template DNA to guide replacement of RNA primer with DNA
* DNA ligase seals the nick by
* hydrolyzing an ATP, to attach to the backbone, then an AMP is released to provide the energy for the nick to be sealed:
* THIS IS A DIFFERENT REACTION THAN WHAT DNA POLYMERASE DOES

Summary of bacterial DNA Replication

Primosome

helicase + primase

Predominant helicase is on which strand?

The lagging strand

How is the leading strand synthesized? How about the lagging strand?

Synthesized continuously from single RNA Primer. Lagging strand is synthesized discontinuously from multiple primers.

Okazaki fragments

RNA primer + DNA

DNA synthesis proceeds (nucleotides are added) in which direction?

5’ → 3’