Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.     

Response of the Bacteriophage T4 Replisome to Noncoding Lesions and Regression of a Stalled Replication Fork

DNA is constantly damaged by endogenous and exogenous agents. The resulting DNA lesions have the potential to halt the progression of the replisome, possibly leading to replication fork collapse. Here, we examine the effect of a noncoding DNA lesion in either leading strand template or lagging strand template on the bacteriophage T4 replisome. A damaged base in the lagging strand template does not affect the progression of the replication fork. Instead, the stalled lagging strand polymerase recycles from the lesion and initiates the synthesis of a new Okazaki fragment upstream of the damaged base. In contrast, when the replisome encounters a blocking lesion in the leading strand template, the replication fork only travels approximately 1 kb beyond the point of the DNA lesion before complete replication fork collapse. The primosome and the lagging strand polymerase remain active during this period, and an Okazaki fragment is synthesized beyond the point of the leading strand lesion. There is no evidence for a new priming event on the leading strand template. Instead, the DNA structure that is produced by the stalled replication fork is a substrate for the DNA repair helicase UvsW. UvsW catalyzes the regression of a stalled replication fork into a “chicken-foot” structure that has been postulated to be an intermediate in an error-free lesion bypass pathway.     

Escherichia coli PriA helicase: fork binding orients the helicase to unwind the lagging strand side of arrested replication forks

Escherichia coli PriA is a primosome assembly protein with 3' to 5' helicase activity whose apparent function is to promote resumption of DNA synthesis following replication-fork arrest. Here, we describe how initiation of helicase activity on DNA forks is influenced by both fork structure and by single-strand DNA-binding protein. PriA could recognize and unwind forked substrates where one or both arms were primarily duplex, and PriA required a small (two bases or larger) single-stranded gap at the fork in order to initiate unwinding. The helicase was most active on substrates with a duplex lagging-strand arm and a single-stranded leading-strand arm. On this substrate, PriA was capable of translocating on either the leading or lagging strands to unwind the duplex ahead of the fork or the lagging-strand duplex, respectively. Fork-specific binding apparently orients the helicase domain to unwind the lagging-strand duplex. Binding of single-strand-binding protein to forked templates could inhibit unwinding of the duplex ahead of the fork but not unwinding of the lagging-strand duplex or translocation on the lagging-strand template. While single-strand-binding protein could inhibit binding of PriA to the minimal, unforked DNA substrates, it could not inhibit PriA binding to forked substrates. In the cell, single-strand-binding protein and fork structure may direct PriA helicase to translocate along the lagging-strand template of forked structures such that the primosome is specifically assembled on that DNA strand.     

Dynamics of Replication Fork Progression Following Helicase–Polymerase Uncoupling in Eukaryotes

Leading-strand polymerase stalling at DNA damage impairs replication fork progression. Using biochemical approaches, we show this arises due to both slower template unwinding following helicase–polymerase uncoupling and establishment of prolonged stalled fork structures. Fork slowing and stalling occur at structurally distinct lesions, are always associated with continued lagging-strand synthesis, are observed when either Pol ε or Pol δ stalls at leading-strand damage, and do not require specific helicase–polymerase coupling factors. Hence, the key trigger for these replisome-intrinsic responses is cessation of leading-strand polymerization, revealing this as a crucial driver of normal replication fork rates. We propose that this helps balance the need for sufficient uncoupling to activate the DNA replication checkpoint with excessive destabilizing single-stranded DNA exposure in eukaryotes.     

Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.     

Functional uncoupling of twin polymerases: mechanism of polymerase dissociation from a lagging-strand block

Replication forks are constantly subjected to events that lead to fork stalling, stopping, or collapse. Using a synthetic rolling circle DNA substrate, we demonstrate that a block to the lagging-strand polymerase does not compromise helicase or leading-strand polymerase activity. In fact, lagging-strand synthesis also continues. Thus, the blocked lagging-strand enzyme quickly dissociates from the block site and resumes synthesis on new primed sites. Furthermore, studies in which the lagging polymerase is continuously blocked show that the leading polymerase continues unabated even as it remains attached to the lagging-strand enzyme. Hence, upon encounter of a block to the lagging stand, the polymerases functionally uncouple yet remain physically associated. Further study reveals that naked single-stranded DNA results in disruption of a stalled polymerase from its beta-DNA substrate. Thus, as the replisome advances, the single-stranded DNA loop that accumulates on the lagging-strand template releases the stalled lagging-strand polymerase from beta after SSB protein is depleted. The lagging-strand polymerase is then free to continue Okazaki fragment production.     

DNA复制研究步入单分子时代

DNA复制是生命体内必不可少的基本过程之一。传统研究显示DNA复制体中前导链和后随链的合成速度总体来说是一致的,从而避免在新生链中产生明显的单链缺口。主流的观点认为这是由于负责前导链和后随链的两个DNA聚合酶分子之间存在着某种协调同步机制。然而,Kowalczykowski实验室最近采用单分子荧光显微技术实时跟踪发现,大肠杆菌DNA复制体前导链和后随链上两个DNA聚合酶分子互相独立工作,并且都不是匀速行进而是呈现断断续续、时快时慢的随机动态变化。当DNA聚合酶暂停复制时,解旋酶仍会持续解链,导致解旋酶和聚合酶短暂的分离。有意思的是,此时DNA复制体触发一种类似“死人键”(dead-man’s switch)的保险机制,使DNA解旋的速度降低80%,从而恢复解旋酶和聚合酶的偶联。基于单分子水平的实时观察,他们认为前导链和后随链DNA复制进程均遵循一个符合高斯分布的随机模型。这与传统的生化研究观察到两者的合成速度总体来说是一致的并不矛盾。Kowalczykowski实验室的研究实现了从复制开始到结束整个过程对每个单分子行为的连续观测,而传统研究反映的则是经过较长时间对多分子群体平均水平的最终结果进行测定。因此,单分子技术可以极大地弥补传统生化研究的不足。随着未来单分子技术的进步和更广泛的应用,必将把包括DNA复制在内的生物学研究带到一个新的时代。     

E. coli DNA replication in the absence of free β clamps

During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the β sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps.     

Polymerase switching in DNA replication

Our view of DNA replication has been of two coupled DNA polymerases anchored to the replication fork helicase in a "replisome" complex, synthesizing leading and lagging strands simultaneously. New evidence suggests that three DNA polymerases can be accommodated into the replisome and that polymerases and repair factors are dynamically recruited and engaged without dismantling of the replisome.     

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer–predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.     

Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement

A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.     

Structure of a human replisome shows the organisation and interactions of a DNA replication machine

The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45‐MCM‐GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading‐strand polymerase Pol ε, together with TIMELESS‐TIPIN, CLASPIN and AND‐1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Å cryo‐EM structure of a human replisome comprising CMG, Pol ε, TIMELESS‐TIPIN, CLASPIN and AND‐1 bound to replication fork DNA. The structure permits a detailed understanding of how AND‐1, TIMELESS‐TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS‐TIPIN with replication fork DNA suggests a mechanism for strand separation.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size.

The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.     

Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae

Mrc1 associates with replication forks, where it transmits replication stress signals and is required for normal replisome pausing in response to nucleotide depletion. Mrc1 also plays a poorly understood role in DNA replication, which appears distinct from its role in checkpoint signaling. Here, we demonstrate that Mrc1 functions constitutively to promote normal replication fork progression. In mrc1Delta cells, replication forks proceed slowly throughout chromatin, rather than being specifically defective in pausing and progression through loci that impede fork progression. Analysis of genetic interactions with Rrm3, a DNA helicase required to resolve paused forks, indicates that Mrc1 checkpoint signaling is dispensable for the resolution of stalled replication forks and suggests that replication forks lacking Mrc1 create DNA damage that must be repaired by Rrm3. These findings elucidate a central role for Mrc1 in normal replisome function, which is distinct from its role as a checkpoint mediator, but nevertheless critical to genome stability.     

The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.     

Timing, Coordination, and Rhythm: Acrobatics at the DNA Replication Fork

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field.     

Human Primpol1: a novel guardian of stalled replication forks

Huang and colleagues identify a human primase-polymerase that is required for stalled replication fork restart and the maintenance of genome integrity.EMBO reports (2013) 14 12, 1104–1112 doi:10.1038/embor.2013.159The successful duplication of genomic DNA during S phase is essential for the proper transmission of genetic information to the next generation of cells. Perturbation of normal DNA replication by extrinsic stimuli or intrinsic stress can result in stalled replication forks, ultimately leading to abnormal chromatin structures and activation of the DNA damage response. On formation of stalled replication forks, many DNA repair and recombination pathway proteins are recruited to resolve the stalled fork and resume proper DNA synthesis. Initiation of replication at sites of stalled forks differs from traditional replication and, therefore, requires specialized proteins to reactivate DNA synthesis. In this issue of EMBO reports, Wan et al [1] introduce human primase-polymerase 1 (hPrimpol1)/CCDC111, a novel factor that is essential for the restart of stalled replication forks. This article is the first, to our knowledge, to ascertain the function of human Primpol enzymes, which were originally identified as members of the archaeao-eukaryotic primase (AEP) family [2].Single-stranded DNA (ssDNA) forms at stalled replication forks because of uncoupling of the DNA helicase from the polymerase, and is coated by replication protein A (RPA) for stabilization and recruitment of proteins involved in DNA repair and restart of replication. To identify novel factors playing important roles in the resolution of stalled replication forks, Wan and colleagues [1] used mass spectrometry to identify RPA-binding partners. Among the proteins identified were those already known to be located at replication forks, including SMARCAL1/HARP, BLM and TIMELESS. In addition they found a novel interactor, the 560aa protein CCDC111. This protein interacts with the carboxyl terminus of RPA1 through its own C-terminal region, and localizes with RPA foci in cells after hydroxyurea or DNA damage induced by ionizing irradiation. Owing to the presence of AEP and zinc-ribbon-like domains at the amino-terminal and C-terminal regions, respectively [2], CCDC111 was predicted to have both primase and polymerase enzymatic activities, which was confirmed with in vitro assays, leading to the name hPrimpol1 for this unique enzyme.The most outstanding discovery in this article is that hPrimpol1 is required for the restart of DNA synthesis from a stalled replication fork (Fig 1). With use of a single DNA fibre assay, knock down of hPrimpol1 had no effect on normal replication-fork progression or the firing of new origins in the presence of replication stress. After removal of replication stress, however, the restart of stalled forks was significantly impaired. Furthermore, the authors observed that hPrimpol1 depletion enhanced the toxicity of replication stress to human cells. Together, these data suggest that hPrimpol1 is a novel guardian protein that ensures the proper re-initiation of DNA replication by control of the repriming and repolymerization of newly synthesized DNA.Open in a separate windowFigure 1The role of hPrimpol1 in stalled replication fork restart. (A) Under normal conditions, the replicative helicase unwinds parental DNA, generating ssDNA that is coated by RPA and serves as a template for leading and lagging strand synthesis. Aside from interacting with RPA bound to the short stretches of ssDNA, the role of hPrimpol1 in normal progression of replication forks is unknown. (B) Following repair of a stalled replication fork, (1) hPrimpol1 rapidly resumes DNA synthesis of long stretches of RPA-coated ssDNA located at the stalled fork site. Later, the leading-strand polymerase (2) or lagging-strand primase and polymerase (3) replace hPrimpol1 to complete replication of genomic DNA. RPA, replication protein A; ssDNA, single-stranded DNA.Eukaryotic DNA replication is initiated at specific sites, called origins, through the help of various proteins, including ORC, CDC6, CDT1 and the MCM helicase complex [3]. On unwinding of the parental duplexed DNA, lagging strand ssDNA is coated by the RPA complex and used as a template for newly synthesized daughter DNA. DNA primase, a type of RNA polymerase, catalyses short RNA primers on the RPA-coated ssDNA that facilitate further DNA synthesis by DNA polymerase. While the use of a short RNA primer is occasionally necessary to restart leading-strand replication, such as in the case of a stalled DNA polymerase, it is primarily utilized in lagging-strand synthesis for the continuous production of Okazaki fragments. The lagging-strand DNA polymerase must efficiently coordinate its action with DNA primase and other replication factors, including DNA helicase and RPA [4]. Cooperation between DNA polymerase and primase is disturbed after DNA damage, ultimately resulting in the collapse of stalled replication forks. Until now, it was believed that DNA primase and DNA polymerase performed separate and catalytically unique functions in replication-fork progression in human cells, but this report provides the first example, to our knowledge, of a single enzyme performing both primase and polymerase functions to restart DNA synthesis at stalled replication forks after DNA damage (Fig 1).… this report provides the first example of a single enzyme performing both primase and polymerase function to restart DNA synthesis at stalled replication forksA stalled replication fork, if not properly resolved, can be extremely detrimental to a cell, causing permanent cell-cycle arrest and, ultimately, death. Therefore, eukaryotic cells have developed many pathways for the identification, repair and restart of stalled forks [5]. RPA recognizes ssDNA at stalled forks and activates the intra-S-phase checkpoint pathway, which involves various signalling proteins, including ATR, ATRIP and CHK1 [6]. This checkpoint pathway halts cell-cycle progression until the stalled forks are properly repaired and restarted. Compared with the recognition and repair of stalled forks, the mechanism of fork restart is relatively elusive. Studies have, however, begun to shed light on this process. For instance, RPA-directed SMARCAL1 has been discovered to be important for restart of DNA replication in bacteria and humans [7]. Together with the identification of hPrimpol1, these findings have helped to expand the knowledge of the mechanism of restarting DNA replication. Furthermore, both reports raise many questions regarding the cooperative mechanism of hPrimpol1 and SMARCAL1 with RPA at stalled forks to ensure genomic stability and proper fork restart [7].First, these findings raise the question of why cells need the specialized hPrimpol1 to restart DNA replication at stalled forks rather than using the already present DNA primase and polymerase. One possibility is that other DNA polymerases are functionally inhibited due to the response of the cell to DNA damage. Although the cells are ready to restart replication, the impaired polymerases might require additional time to recover after DNA damage, necessitating the use of hPrimpol1. In support of this idea, we found that the p12 subunit of DNA polymerase δ is degraded by CRL4^CDT2 E3 ligase after ultraviolet damage [8]. As a result, alternative polymerases, such as hPrimpol1, could compensate for temporarily non-functioning traditional polymerases. A second explanation is that the polymerase and helicase uncoupling after stalling of a fork results in long stretches of ssDNA that are coated with RPA. To restart DNA synthesis, cells must quickly reprime and polymerize large stretches of ssDNA to prevent renewed fork collapse. By its constant interaction with RPA1, hPrimpol1 is present on the ssDNA and can rapidly synthesize the new strand of DNA after the recovery of stalled forks. Third, the authors found that the association of hPrimpol1 with RPA1 is independent of its functional AEP and zinc-ribbon-like domains and occurs in the absence of DNA damage. These results might indicate a role for hPrimpol1 in normal replication fork progression, but further work is necessary to determine whether that is true.The discovery of hPrimpol1 is also important in an evolutionary contextSeveral questions remain. First, what is the fidelity of the polymerase activity? Other specialized polymerases that act at DNA damage sites sometimes have the ability to misincorporate a nucleotide across from a site of damage, for example pol-eta and -zeta [9]. It will be interesting to know whether hPrimpol1 is a high-fidelity polymerase or an error-prone polymerase. Second, is the polymerase only brought into action after fork stalling? If hPrimpol1 is an error-prone polymerase, one could envision other types of DNA damage that can be bypassed by hPrimpol1. Third, is the primase selective for ribonucleotides, or can it also incorporate deoxynucleotides? The requirement of the same domain—AEP—for primase and polymerase activities raises the possibility that NTPs or dNTPs could be used for primase or polymerase activities.The discovery of hPrimpol1 is also important in an evolutionary context. In 2003, an enzyme with catalytic activities like that of hPrimpol1 was discovered in a thermophilic archeaon and in Gram-positive bacteria [10]. This protein had several catalytic activities in vitro, including ATPase, primase and polymerase. In contrast to these Primpol enzymes, those capable of primase and polymerase functions had not been found in higher eukaryotes, which suggested that evolutionary pressures forced a split of these dual-function enzymes. Huang et al''s report suggests, however, that human cells do in fact retain enzymes similar to Primpol. In summary, the role of hPrimpol1 at stalled forks broadens our knowledge of the restart of DNA replication in human cells after fork stalling, allowing for proper duplication of genomic DNA, and provides insight into the evolution of primases in eukaryotes.     

The DNA repair helicase UvrD is essential for replication fork reversal in replication mutants

Replication forks arrested by inactivation of the main Escherichia coli DNA polymerase (polymerase III) are reversed by the annealing of newly synthesized leading- and lagging-strand ends. Reversed forks are reset by the action of RecBC on the DNA double-strand end, and in the absence of RecBC chromosomes are linearized by the Holliday junction resolvase RuvABC. We report here that the UvrD helicase is essential for RuvABC-dependent chromosome linearization in E. coli polymerase III mutants, whereas its partners in DNA repair (UvrA/B and MutL/S) are not. We conclude that UvrD participates in replication fork reversal in E. coli.     

PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates microreview

The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D-loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3' to 5' helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA's repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. IV. Reconstitution of an asymmetric, dimeric DNA polymerase III holoenzyme.

Individually purified subunits have been used to reconstitute the action of the Escherichia coli DNA polymerase III holoenzyme (Pol III HE) at a replication fork formed in the presence of the primosome, the single-stranded DNA binding protein, and a tailed form II DNA template. Complete activity, indistinguishable from that of the intact DNA Pol III HE, could be reproduced with a combination of the DNA polymerase III core (Pol III core), the gamma.delta complex, and the beta subunit. Experiments where the Pol III core in reaction mixtures containing active replication forks was diluted suggested that the lagging-strand Pol III core remained associated continuously with the replication fork through multiple cycles of Okazaki fragment synthesis. Since the lagging-strand Pol III core must dissociate from the 3' end of the completed Okazaki fragment, this suggests that its association with the fork is via protein-protein interactions, lending credence to the idea that it forms a dimeric complex with the leading-strand Pol III core. An asymmetry in the action of the subunits was revealed under conditions (high ionic strength) that were presumably destabilizing to the integrity of the replication fork. Under these conditions, tau acted to stimulate DNA synthesis only when the primase was present (i.e. when lagging-strand DNA synthesis was ongoing). This stimulation was reflected by an inhibition of the formation of small Okazaki fragments, suggesting that, within the context of the model developed to account for the temporal order of steps during a cycle of Okazaki fragment synthesis, the presence of tau accelerated the transit of the lagging-strand Pol III core from the 3' end of the completed Okazaki fragment to the 3' end of the new primer.