8 pts - Discuss how an E. coli origin of replication is recognized [sequences?], opened [by DnaA], and helicase is added – include any important sequences and their roles in the story. How is this origin licensed?

Ans:

Initiation of replication in E coli requires a sequential congregation of six proteins DnaA, DnaB, DnaC, HU, gyrase, and SSB. DnaA is the first origin recognition protein which is the “licensing factor”. There are specific sequences for DnaA in the oriC called “DnaA boxes” to which it binds. DnaA box is a nine base pair repeat sequence motif and five such DNA boxes (R1 to R5) are present in the E. coli oriC. During most of the cell cycle, the DnaA is bound to R1, R2, and R4 but just before initiation, it binds to R3 and R5, after binding to ATP. DnaA also has an intrinsic ATPase activity that is required for timing such that there is no re-initiation. In addition to this, near to the left border of the oriC there are three 13 mer repeat motifs also. Together these 9 mer and 13 mer sequences define the minimal 245bp origin of replication.

Now in addition to the transcription of the licensing factor either of the two genes flanking the E.coli. oriC must be transcribed and there must be cell wall synthesis going on. This aids in forming the replication bubble. First, the DnaA-ATP binds to the fully methylated oriC sequence motif (9 mer as stated above) aided by the accessory protein DiaA on the right side of the oriC. Then it binds to the 13 mer sequences on the left side of the oriC. During this, the DnaA transforms from an extended form to the compact form which causes the bending of DNA and opens the double helix. Following this, the DiaA is removed and two priming complex of DnaB and DnaC-ATP are recruited. DnaB is in the hexameric state and DnaC is six monomers. As soon as the complex binds to the single-stranded DNA, the DnaC hydrolyzes the ATP, and DnaC is removed, and DnaB is activated. DnaB displaces DnaA from the 13 mer sequence and extends the opening of DNA. In this way, the region of unwinding is extended. DnaB also activates DnaG primase in both the stranded. Gyrase provides a swivel around which the DNA strand can rotate around the other strand (so that twisting is not there) and SSB proteins bind to the single strand to stabilize them so that they are not again twisted and closed.

12 pts - Discuss the roles of the various enzymes and proteins in DNA replication at an open replication fork in E. coli. Make sure you compare and contrast the leading and lagging strand processes, and the functionality of the different enzymes and proteins used.

• topoisomerase,

Ans: Topoisomerase II or gyrase is an enzyme that is involved in helping the DNA strand revolve around the other to relax their winding. During DNA replication as the helicase unwinds the DNA and replication bubble is formed, and then at the two borders of that replication bubble the DNA strands face a torsional strain due to over-winding. To ease that Topoisomerase provides a swivel around which once DNA strand movers around the other to relax the tension.

1. You are performing a cation-exchange chromatography experiment o...