The dynamic processivity of the T4 DNA polymerase during replication

Abstract

The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of Ϸ0.0013 sec ؊1 was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution͞replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.DNA replication ͉ polymerase processivity ͉ polymerase exchange B acteriophage T4 DNA polymerase is responsible for DNA synthesis on both leading and lagging strands. This enzyme is the gene 43 product (gp43), which, along with seven other T4 replication proteins, constitutes the T4 replisome that carries out coordinated DNA synthesis (1). Among these seven proteins, the clamp loader (gp44͞62) and the clamp protein (gp45) are polymerase accessory factors that significantly increase the processivity of gp43 during replication by forming the holoenzyme complex (2). The current working model of T4 DNA replication involves two such holoenzyme complexes acting on leading and lagging strands (3). Moving ahead of the holoenzyme complexes is the T4 primosome generated from the helicase (gp41), the primase (gp61), and the helicase accessory protein (gp59). This unit is required to rapidly unwind the double-stranded DNA in front of the moving fork (4, 5) and to synthesize the pentaribonucleotide primers for Okazaki fragment synthesis (6). The last replisome component is the singlestranded DNA (ssDNA) binding protein (gp32) which is involved in the stabilization of the ssDNA loop structure generated during lagging strand synthesis (7) and in the organization of the replisome (8, 9).The entire 172-kb T4 genome is fully duplicated within Ϸ15 min (10). As a result, the high processivity of the polymerase is crucial for efficient DNA replication. The holoenzyme stability has been measured on a short, defined DNA fork to give a half-life of Ϸ6 min (11). This value is within the same range as the 11-min half-life for the T4 helicase on a moving fork determined by Alberts and...

Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

“…Previously, an active exchange process was observed for the T4 Pol holoenzyme (9). To test whether a similar Pol exchange occurs for yeast Pol␦ we constructed a Pol␦ trap (Pol␦ AA ) by mutating the two active-site aspartates of the catalytic subunit of Pol␦ to alanine (Pol3 D762A, D764A).…”

“…Using a catalytically impaired T4 replicative Pol (gp43) trap, it was found that gp43 from solution undergoes active exchange with gp43 in the holoenzyme (9). A model was proposed to explain the Pol exchange process in T4 phage based on known x-ray crystal structures of both gp43 and gp45.…”

Templating efficiency of naked DNA

“…These reactions occur with high fidelity (1,4). Polymerases also achieve high processivity on leading and lagging strands, despite differences in rate for individual substrates/ templates (5). The mechanism of enzymatic primer extension is complex.…”

T4 replication: What does “processivity” really mean?

“…It is well established that lagging strand components, other than the polymerase, are recruited continuously from solution. The current study by Yang et al (2) shows that the polymerase is exchangeable within the replisome complex but only rarely leaves the complex if a replacement polymerase is not available. The helicase appears to be the most solidly fixed component and has been suggested to be the ''cornerstone'' of the replisome (1).…”

“…The most obvious is in replication past a blocking lesion on the template. The exchange mechanism proposed by Yang et al (2) provides a means of transferring the primer terminus from a stalled replicative polymerase to a specialized lesion bypass polymerase. One can envisage a straightforward targeting process.…”