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Replication of a bacterial chromosome starts at a specific point, the origin of replication.

14). This property of Pol I is used in the laboratory for labeling small fragments of DNA with radioactive nucleotides by a procedure known as "nick translation." Nicks are introduced into one strand of the DNA by DNaseI (deoxyribonuclease I). DNA polymerase I then starts at the nick and moves along the DNA removing the nucleotides ahead of it and replacing them with the radioactive nucleotides provided. Both DNA polymerase I and DNA ligase have other important uses in genetic engineering (see Ch. 22).

DNA polymerases I and II were discovered before DNA polymerase III, hence the numbering. In retrospect, it is easy to understand why Pol III, with its complex requirements and multiple subunits, took longer to discover than the relatively simple enzymes Pol I and Pol II which are involved in DNA repair.

Chromosome Replication Initiates at oriC

So far we have discussed the processes involved in the duplication of the DNA double helix. In addition, DNA replication must be synchronized with cell division. This involves starting replication at a specific location on the chromosome and stopping when the chromosome has been successfully copied. Prokaryotic DNA replication starts at a unique site on the chromosome, known as the origin of replication, and proceeds in both directions around the circle. The initiation complex contains five proteins: DnaA, DnaB, DnaC, gyrase and SSB. Of these, only DnaA is unique to chromosome initiation; the others are also involved in starting new Okazaki fragments. The origin, oriC, has three 13-base repeats, each consisting of GATCTNTTNTTTT, followed by four nine-base repeats, each consisting of TTATNCANA. Note that both sequences are AT-rich, a feature which aids strand separation. These are scattered over a 245 base pair region that is required for chromosome initiation (Fig. 5.18).

The first event is binding of DnaA protein to the four nine-base sequences. A cluster of 30 or so DnaA proteins is bound and the whole oriC region is wrapped around them. Next, the DnaA proteins open the DNA at all three of the 13-base repeats. Next to join is the DnaB helicase. Each hexamer of DnaB is at first accompanied by six DnaC proteins, needed to load DnaB onto the DNA. DnaB displaces DnaA from the single stranded 13-base repeats and begins to unwind the DNA and so create a replication fork (Fig. 5.19).A second DnaB hexamer creates a second replication fork moving in the opposite direction. DNA gyrase is also needed to allow unwinding and SSB proteins are needed to keep the DNA single stranded. DnaB also activates the primase, which then makes an RNA primer at each of the two points where the two leading strands are initiated.

Plasmids have been constructed in the laboratory that carry the chromosomal origin sequences of E. coli. This has allowed analysis of initiation in a cell-free system in which individual proteins are added to a DNA molecule of manageable size. The binding of the DnaA/DnaB/DnaC complex to oriC has been seen under the electron microscope (Fig. 5.20).

DnaA protein Protein that binds to the origin of bacterial chromosomes and helps initiate replication initiation complex (for replication) Assemblage of proteins that binds to the origin and initiates replication of DNA

nick translation The removal of a short stretch of DNA or RNA, starting from a nick, and its replacement by newly made DNA

origin of replication Site on a DNA molecule where replication begins

Chromosome Replication Initiates at oriC 119

A) DnaA - DNA AGGREGATES

B) Replication bubbles forms

C) DnaB AND DnaC BIND TO FORM

REPLICATION FORKS AND DISPLACE DnaA

Helicase Helicase

DnaB DnaB

yjk. jy

UM/W

DnaA

DnaA

DnaA

DnaC

FIGURE 5.19 Three Steps in the Initiation of Replication by DnaA

A) DnaA protein binds first to the four nine-base repeats, and then to the three 13-base repeats. B) As more DnaA binds, the DNA folds and the three 13-base repeats are unwound. C) Two complexes of DnaB and DnaC bind to the three 13-base repeats. This pushes DnaA away and causes the DNA strand to open all along the AT-rich region. The two DnaB complexes now start two replication forks, each headed in opposite directions around the circular DNA.

FIGURE 5.20 Initiation Complex Bound to oriC

The binding of the DnaA/DnaB/DnaC complex to oriC has been seen under the electron microscope. A plasmid carrying the oriC region was used instead of chromosomal DNA. Complexes were formed on supercoiled DNA of plasmid pCM959 with the proteins DnaA, DnaB, DnaC, plus HU. The complexes were cross-linked and the plasmid DNA was cut with Ban1 to produce six fragments. The origin, oriC, was asymmetrically situated on the 703-bp fragment shown here. From: Funnell, Baker and Kornberg, In vitro assembly of a pre-priming complex at the origin of the Escherichia coli chromosome. Journal of Biological Chemistry, 262 (1987) 10327-10334.

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