DNA Replication

Mechanism of DNA translocation in a replicative

helicase

• Unwind dsDNA to make ssDNA– DNA replication– Repair– Recombination

Helicase Function

• E1 & TAG (virus)– Recognize & melts

origin to create ssDNA

– Assemble on ssDNA– E1 3’-5’ to Unwind

Helicase Function

• Crystal structure of hexameric helicase (E1 of bovine papilomavirus)

• Shows the path of DNA• Indicates mechanism

Crystalization Conditions

• Fragment BPV-1 E1 – Lacks N-term (306-577)– GST fusion

• Concentrated with– 5 mM ADP– 200 mM MgCl2– Oligo T (13 mer)

– Mother liquor 100 mM KCl– Crystals

• Flash frozen (N2 (lq))• Synchotron Radiation

– Molecular replacement

http://en.wikipedia.org/wiki/Image:X_ray_diffraction.png

5’ 3’

Courtesy of Chayne Piscitelli

Loops

roll

D504, R505 & K508

Staircase Model

 A helicase staircase. Raney KD. Nat Struct Mol Biol. 2006 Aug;13(8):671-2.

ATP?

Y534

ADP

ATP

Escort Model