Genes VII

Following generation of a replication fork as indicated in Figure 13.26, the priming reaction occurs to generate a leading strand. We know that synthesis of RNA is used for the priming event, but the details of the reaction are not known. Some mutations in dnaA can be suppressed by mutations in RNA polymerase, which suggests that DnaA could be involved in an initiation step requiring RNA synthesis in vivo.

RNA polymerase could be required to read into the origin from adjacent transcription units; by terminating at sites in the origin, it could provide the 3′ VOH ends that prime DNA polymerase III. (An example is provided by the use of D loops at mitochondrial origins, as discussed in 12 The replicon.) Alternatively, the act of transcription could be associated with a structural change that assists initiation. This latter idea is supported by observations that transcription does not have to proceed into the origin; it is effective up to 200 bp away from the origin, and can use either strand of DNA as template in vitro. The transcriptional event is inversely related to the requirement for supercoiling in vitro, which suggests that it acts by changing the local DNA structure so as to aid melting of DNA.

Another system for investigating interactions at the origin is provided by phage lambda, whose origin sponsors bidirectional replication. A map of the region is shown in Figure 13.28. Initiation of replication at the lambda origin requires "activation" by transcription starting from P_R. As with the events at oriC, this does not necessarily imply that the RNA provides a primer for the leading strand. Analogies between the systems suggest that RNA synthesis could be involved in promoting some structural change in the region.

Initiation requires the products of phage genes O and P, as well as several host functions. The phage O protein binds to the lambda origin; the phage P protein interacts with the O protein and with the bacterial proteins. The origin lies within gene O, so the protein acts close to its site of synthesis.

Variants of the phage called λdv consist of shorter genomes that carry all the information needed to replicate, but lack infective functions. λdv DNA survives in the bacterium as a plasmid, and can be replicated in vitro by a system consisting of the phage-coded proteins O and P together with bacterial replication functions.

Lambda proteins O and P form a complex together with DnaB at the lambda origin, oriλ. The origin consists of two regions; as illustrated in Figure 13.29, a series of four binding sites for the O protein is adjacent to an A PT-rich region.

The first stage in initiation is the binding of O to generate a roughly spherical structure of diameter ~11 nm, sometimes called the O-some. The O-some contains ~100 bp or 60 kD of DNA. There are four 18 bp binding sites for O protein, which is ~34 kD. Each site is palindromic, and probably binds a symmetrical O dimer. The DNA sequences of the O-binding sites appear to be bent, and binding of O protein induces further bending.

If the DNA is supercoiled, binding of O protein causes a structural change in the origin. The A PT-rich region immediately adjacent to the O-binding sites becomes susceptible to S1 nuclease, an enzyme that specifically recognizes unpaired DNA. This suggests that a melting reaction occurs next to the complex of O proteins.

The role of the O protein is analogous to that of DnaA at oriC: it prepares the origin for binding of DnaB. Lambda provides its own protein, P, which substitutes for DnaC, and brings DnaB to the origin. When lambda P protein and bacterial DnaB proteins are added, the complex becomes larger and asymmetrical. It includes more DNA (a total of ~160 bp) as well as extra proteins. The λ P protein has a special role: it inhibits the helicase action of DnaB. Replication fork movement is triggered when P protein is released from the complex. Priming and DNA synthesis follow.

Some proteins are essential for replication without being directly involved in DNA synthesis as such. Interesting examples are provided by the DnaK and DnaJ proteins. DnaK is a chaperone, related to a common stress protein of eukaryotes. Its ability to interact with other proteins in a conformation-dependent manner plays a role in many cellular activities, including replication. The role of DnaK/DnaJ may be to disassemble the pre-priming complex; by causing the release of P protein, they allow replication to begin.

The initiation reactions at oriC and oriλ are similar. The same stages are involved, and rely upon overlapping components. The first step is recognition of the origin by a protein that binds to form a complex with the DNA, DnaA for oriC and O protein for oriλ. A short region of A PT-rich DNA is melted. Then DnaB is loaded; this requires different functions at oriC and oriλ (and yet other proteins are required for this stage at other origins). When the helicase DnaB joins the complex, a replication fork is created. Finally an RNA primer is synthesized, after which replication begins.

The use of oriC and oriλ provides a general model for activation of origins. A similar series of events occurs at the origin of the virus SV40 in mammalian cells. Two hexamers of T antigen, a protein coded by the virus, bind to a series of repeated sites in DNA. In the presence of ATP, changes in DNA structure occur, culminating in a melting reaction. In the case of SV40, the melted region is rather short and is not A PT-rich, but it has an unusual composition in which one strand consists almost exclusively of pyrimidines and the other of purines. Near this site is another essential region, consisting of A PT base pairs, at which the DNA is bent; it is underwound by the binding of T antigen. An interesting difference from the prokaryotic systems is that T antigen itself possesses the helicase activity needed to extend unwinding, so that an equivalent for DnaB is not needed.