DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.     

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.     

Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.     

Single-molecule analysis of DNA replication in Xenopus egg extracts

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.     

The CMG DNA helicase and the core replisome

Eukaryotic DNA replication is performed by the replisome, a large and dynamic multi-protein machine endowed with the required enzymatic components for the synthesis of new DNA. Recent cryo-electron microscopy (cryoEM) analyses have revealed the conserved architecture of the core eukaryotic replisome, comprising the CMG (Cdc45-MCM-GINS) DNA helicase, the leading-strand DNA polymerase epsilon, the Timeless-Tipin heterodimer, the hub protein AND-1 and the checkpoint protein Claspin. These results bid well for arriving soon at an integrated understanding of the structural basis of semi-discontinuous DNA replication. They further set the scene for the characterisation of the mechanisms that interface DNA synthesis with concurrent processes such as DNA repair, propagation of chromatin structure and establishment of sister chromatid cohesion.     

Dynamic Exchange of Two Essential DNA Polymerases during Replication and after Fork Arrest

The replisome is a multiprotein machine responsible for the faithful replication of chromosomal and plasmid DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in live Bacillus subtilis cells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following replication fork stress using damage-independent and damage-dependent conditions. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replication proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results suggest that DnaE provides a limited contribution to chromosomal replication and repair in vivo.     

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.     

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。     

Spatial and temporal organization of the Bacillus subtilis replication cycle

DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.     

A hand-off mechanism for primosome assembly in replication restart

Collapsed DNA replication forks must be reactivated through origin-independent reloading of the replication machinery (replisome) to ensure complete duplication of cellular genomes. In E. coli, the PriA-dependent pathway is the major replication restart mechanism and requires primosome proteins PriA, PriB, and DnaT for replisome reloading. However, the molecular mechanisms that regulate origin-independent replisome loading are not fully understood. Here, we demonstrate that assembly of primosome protein complexes represents a key regulatory mechanism, as inherently weak PriA-PriB and PriB-DnaT interactions are strongly stimulated by single-stranded DNA. Furthermore, the binding site on PriB for single-stranded DNA partially overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which single-stranded DNA is handed off from one primosome protein to another as a repaired replication fork is reactivated. This model helps explain how origin-independent initiation of DNA replication is restricted to repaired replication forks, preventing overreplication of the genome.     

The many facets of the Tim-Tipin protein families’ roles in chromosome biology

Failures in DNA replication are a potent force for driving genome instability. The proteins which form the replisome, the DNA replication machinery, play a fundamental role in preventing replicative catastrophes. The Tim (TIMELESS/TIMEOUT) and Tipin proteins are two conserved replisome associated proteins which have functions in preventing replication fork collapse and replicative checkpoint signalling in response to factors which slow the progression of the replisome. Intriguingly, TIMELESS family members have been implicated in the regulation of the biological clock, giving a tantalising pointer to a possible link between DNA replication and circadian rhythm control. Here we report on our current understanding of the many facets of these protein families in maintaining genome stability and replication checkpoint control.     

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.     

Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases

The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.     

Chromosome organization and segregation in bacteria

In the recent years, considerable advances have been made towards understanding the structure and function of the bacterial chromosome. A number of different factors appear to cooperate in condensing DNA into a highly dynamic assembly of supercoiled loops. Despite this variability in the lower levels of chromatin structure, the global arrangement of chromosomal DNA within the cell is surprisingly conserved, with loci being arrayed along the cellular long axis in line with their order on the genomic map. This conserved pattern is propagated during the course of DNA segregation. First, after entry into S-phase, the newly synthesized origin regions are segregated in an active and directed process, involving the bacterial actin homolog MreB. Subsequent DNA segments then follow by different mechanisms. They are separated immediately after release from the replisome and move rapidly to their conserved positions in the incipient daughter cell compartments. Partitioning of the bacterial chromosome thus takes place while DNA replication is in progress.     

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.     

Replication and recombination of herpes simplex virus DNA

Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.