Single Molecule Analysis of differential functional mechanisms of MtRecG while being exposed to variants of stalled replication-fork

Helicases are motor proteins involved in multiple activities to carry out manipulation of the nucleic acids for efficient gene regulation. In case of roadblocks that can lead the replication machinery to get halted, a complex molecular surveillance system utilizing helicases as its key player ensures the halted fork to resume its duplication process. RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. Here, through adoption of single-molecule techniques we have attempted to probe the DNA unwinding features by RecG and tried to capture several stages of genetic rearrangement. An elevated processivity of RecG has been observed for the kinds of stalled fork where progression of lagging daughter strand is ahead than that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. In summary, we have featured that RecG adopts asymmetric mode of locomotion to unwind the lagging daughter strand to facilitate Holliday junction creation which acts as a suitable intermediate for recombinational repair pathway.

Global landscape of replicative DNA polymerase usage in the human genome

The division of labour between DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combing the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality, zones of replication initiation and termination. We confirm that transcriptional activity shapes the patterns of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co-directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

DNA in Motion

Students often struggle to understand the full implications of some basic chemical concepts of DNA structure and function, especially how DNA’s directionality and antiparallel nature determine key functional features of replication and molecular recombination. Visualizing the complexities of these processes requires a working knowledge of how DNA’s nucleotides are assembled and how these components interact. This article describes a simple activity that can be used to visualize how nucleotides join together, how base pairs form, and, most importantly, how the active processes of replication and recombination are related to DNA chemistry. In this activity, students model DNA structure, with each student representing a single nucleotide, then join together to form a polynucleotide with 5′ to 3′ directionality. Two chains then pair to form the antiparallel DNA duplex. The activity not only illustrates the basic chemistry of DNA but also allows students to participate in active modeling of leading-strand and lagging-strand replication and in the formation of the Holliday junction molecule, the basic intermediate of recombination events including crossing over and gene conversion. The demonstrations can be videotaped from above to make a permanent copy of these events for teaching and study purposes. Example illustrations and links to videos are included.

Bypass of complex co-directional replication-transcription collisions by replisome skipping

Collisions between the replisome and RNA polymerases [RNAP(s)] are the main obstacle to DNA replication. These collisions can occur either head-on or co-directionally with respect to the direction of translocation of both complexes. Whereas head-on collisions require additional factors to be resolved, co-directional collisions are thought to be overcome by the replisome itself using the mRNA transcript as a primer. We show that mRNA takeover is utilized primarily after collisions with single RNAP complexes with short transcripts. Bypass of more complex transcription complexes requires the synthesis of a new primer downstream of the RNAP for the replisome to resume leading-strand synthesis. In both cases, bypass proceeds with displacement of the RNAP. Rep, Mfd, UvrD and RNase H can process the RNAP block and facilitate replisome bypass by promoting the formation of continuous leading strands. Bypass of co-directional RNAP(s) and/or R-loops is determined largely by the length of the obstacle that the replisome needs to traverse: R-loops are about equally as potent obstacles as RNAP arrays if they occupy the same length of the DNA template.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

The E. coli helicase does not use ATP during replication.

The replisome is responsible for replication of DNA in all domains of life, with several of its individual enzyme components relying on hydrolysis of nucleoside triphosphates to provide energy for replisome function. Half a century of biochemical studies have demonstrated a dependence on ATP as an energy source for helicases to unwind duplex DNA during replication. Through single-molecule visualization of DNA replication by the Escherichia coli replisome, we demonstrate that the DnaB helicase does not rely on hydrolysis of ATP (or any ribo-NTPs) in the context of the elongating replisome. We establish that nucleotide incorporation by the leading-strand polymerase is the main motor driving the replication process.

The Mechanism of Replication Stalling and Recovery within Repetitive DNA

SUMMARYAccurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstituted replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We found that structure-prone repeats are sufficient to impair replication. Whilst template unwinding was unaffected, leading strand synthesis was inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats relied on replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats required an extrinsic accessory helicase. DNA-induced fork stalling was mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress also applies to stalling induced by repetitive sequences.