Creating the replication fork

At least 30 different proteins are required to ini-tiate replication and to replicate the DNA in E.

coli. For more information, the student should consult refs. 4 through 11.

DNA replication takes place in a complex DNA-synthesizing “factory” called a repli-some, which consists of many enzymes and proteins that will soon be described. Within the replisome there are replication forks cre-ated on the DNA, and this is where replication takes place. (Clearly, DNA synthesis must be related to the growth rate of the cell. Review Section 2.2.3 for a discussion of the relation-ship between the timing of initiation of DNA replication and the growth rate.) So how is the replication fork made? To make the replica-tion fork, the DNA strands must fi rst unwind

so that each strand can act as a template.

However, the unwinding does not begin at just any place in the DNA, but rather at a particu-lar site in the DNA duplex termed the origin, which should be remembered as the oriC locus.

When the duplex is unwound, a Y is created.

The arms of the Y are single stranded because the duplex has become unwound there; but the region downstream of the juncture, where the two arms come together, is still double stranded (Fig. 3.7). The juncture should be remembered as the replication fork. DNA is usually repli-cated bidirectionally; that is, there are actu-ally two replication forks (Fig. 3.8). (Note 12 explains how directionality can be determined.) Bidirectional replication, which halves the time needed to replicate the DNA molecule, gener-ally takes place with phages, plasmids, bacteria, and eukaryotic cells. The replication forks are thought to be stationary, and the unreplicated DNA appears to thread through them. (This is discussed in Section 3.1.4.) The detailed steps in the creation of a replication fork are described next. Before DNA synthesis can begin, a pre-priming complex must form. The fi rst step is the formation of an “open complex,” which devel-ops into the “prepriming complex.”

Unwinding the duplex: Creation of the prepriming complex

The prepriming complex is formed fi rst and it is created in two stages.⁹ In stage 1 the open Fig. 3.4 Supercoiled DNA. (A) When there are 10.5 base pairs per helical turn, the DNA is relaxed. Native DNA is generally underwound (i.e., >10.5 base pairs per helical turn). Underwinding introduces a strain in the molecule; and to reduce the strain, the molecule twists upon itself to form supercoils (B). Supercoiling result-ing from underwound DNA is referred to as negative supercoilresult-ing. Supercoils will also form if DNA is over-wound (i.e., >10.5 base pairs per turn). Supercoiling due to overover-wound DNA is called positive supercoiling. In negative supercoiling the DNA is twisted in a direction opposite to that of the right-handed double helix, and in positive supercoiling the DNA is twisted in the same direction as the right-handed double helix.

Fig. 3.5 Supercoiling ahead of the replication fork. As the template strands in the closed circle are pulled apart, the duplex ahead of the replication fork overwinds as positive supercoils are formed. The twists in the overwound regions are removed by DNA gyrase, which produces negative supercoils and underwinds the duplex.

complex is formed, and in stage 2 the open complex develops into the prepriming complex.

DNA synthesis, which will be described later, actually begins with the prepriming complex.

1. Formation of the open complex

Creation of the open complex is initiated at the origin of replication (oriC) with ATP and two DNA-binding proteins: DnaA and a his-tonelike protein called HU (Fig. 3.9). See the subsection entitled DNA-binding proteins

infl uence nucleoid structure and gene expres-sion in Section 1.2.6. Within the origin (oriC) there are multiple sites to which DnaA binds.

Approximately 30 DnaA molecules bind to the sites as the DNA wraps around a core of DnaA molecules. Then, in an ATP-dependent reaction that is aided by HU, the adjacent A–T-rich region at the 5′ end of the origin sequence unwinds to form the 45 bp open complex.

However, something must be done to prevent the single strands from coming together again

Fig. 3.6 The action of DNA gyrase: converting positive supercoils into negative supercoils. (A) A positively supercoiled node; that is, the duplex is twisted around its central axis. For example, this happens during DNA replication as the duplex is being unwound by helicase at the replication fork and the duplex ahead of the rep-lication fork spontaneously becomes overwound by being twisted in a clockwise direction. (B) Both strands are cut by DNA gyrase, and then the gyrase passes the uncut portion through the gap and the gap is sealed. (C) The duplex is now twisted in the opposite direction; that is, it is negative supercoiled and underwound. DNA gyrase, an ATP-dependent enzyme, is sometimes referred to as providing a “swivel” that allows the replica-tion fork to continue.

Fig. 3.7. A replication fork. The two template strands on the left have become unwound and are being copied.

The unwinding is caused by DnaB protein, which is the replication fork helicase (not shown) and requires ATP. Note that there are single-stranded regions near the fork. A DNA-binding protein, called SSB (not shown), binds to the single-stranded regions, preventing them from coming together. Now (right) the duplex has both unwound and double-stranded components.

to re-form the duplex. This task is accomplished by single-stranded binding (SSB) proteins that coat the strands.

2. Formation of the prepriming complex The open complex unwinds into the preprim-ing complex. The unwindpreprim-ing is performed by a protein called helicase (DnaB), which must fi rst

bind to the DNA. However, DnaB does not bind to the DNA on its own but must be transferred to the open complex from a DnaB:DnaC:ATP complex. After binding to the DNA, DnaB further unwinds the strands bidirectionally to form the prepriming complex, with two rep-lication forks ready for the initiation of DNA replication.

Timing of initiation

Precisely what determines the timing of ini-tiation of bacterial DNA replication is still a matter of speculation. As discussed in Section 2.2.3, the cell mass per chromosomal replica-tion origin (as opposed to plasmid replicareplica-tion origins) at the time of initiation is constant, and all oriC origins (even plasmid origins), begin replication at the same time. This mass is called the initiation mass or the initiation volume, and some have suggested that it cor-responds to the activity of DnaA. Another question is, what prevents the newly repli-cated origins from reinitiating in the same cell cycle? For a discussion of what regulates the initiation of DNA replication at oriC, see note 13.

3.1.4 Replicating the DNA