Replisomal coupling between the α-Pol III core and the τ-subunit of the clamp loader complex (CLC) are essential for genomic integrity in E. coli.

Coupling interactions between the alpha (α) subunit of the polymerase III core (α-Pol III core) and the tau (τ) subunit of the clamp loader complex (τ-CLC) are vital for efficient and rapid DNA replication in Escherichia coli (E. coli). Specific and targeted mutations in the C-terminal τ-interaction region of the Pol III α-subunit disrupted efficient coupled rolling circle DNA synthesis in vitro and caused significant genomic defects in CRISPR-Cas9 dnaE edited cell strains.

Tau-mediated coupling between Pol III synthesis and DnaB helicase unwinding helps maintain genomic stability.

The τ-subunit of the clamp loader complex physically interacts with both the DnaB helicase and the polymerase III (Pol III) core α-subunit through domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the dnaX gene at the C terminus of τ predicted to interact with the α-subunit of Pol III.

Dysregulated DnaB unwinding induces replisome decoupling and daughter strand gaps that are countered by RecA polymerization.

The replicative helicase, DnaB, is a central component of the replisome and unwinds duplex DNA coupled with immediate template-dependent DNA synthesis by the polymerase, Pol III. The rate of helicase unwinding is dynamically regulated through structural transitions in the DnaB hexamer between dilated and constricted states. Site-specific mutations in DnaB enforce a faster more constricted conformation that dysregulates unwinding dynamics, causing replisome decoupling that generates excess ssDNA and induces severe cellular stress.

Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity.

The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCAT) and several EcCAT:ligand-bound complexes.

Activity, substrate preference and structure of the HsMCM8/9 helicase.

The minichromosomal maintenance proteins, MCM8 and MCM9, are more recent evolutionary additions to the MCM family, only cooccurring in selected higher eukaryotes. Mutations in these genes are directly linked to ovarian insufficiency, infertility, and several cancers. MCM8/9 appears to have ancillary roles in fork progression and recombination of broken replication forks.

A multi-functional role for the MCM8/9 helicase complex in maintaining fork integrity during replication stress.

The minichromosome maintenance (MCM) 8/9 helicase is a AAA complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, a precise cellular role for MCM8/9 has remained elusive. We have interrogated the DNA synthesis ability and replication fork stability in cells lacking MCM8 or 9 and find that there is a functional partitioning of MCM8/9 activity between promoting replication fork progression and protecting persistently stalled forks.

In vivo fluorescent TUNEL detection of single stranded DNA gaps and breaks induced by dnaB helicase mutants in Escherichia coli.

The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress.

Beyond the Lesion: Back to High Fidelity DNA Synthesis.

High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases.

Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness.

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB.

Determining translocation orientations of nucleic acid helicases.

Helicase enzymes translocate along an RNA or DNA template with a defined polarity to unwind, separate, or remodel duplex strands for a variety of genome maintenance processes. Helicase mutations are commonly associated with a variety of diseases including aging, cancer, and neurodegeneration. Biochemical characterization of these enzymes has provided a wealth of information on the kinetics of unwinding and substrate preferences, and several high-resolution structures of helicases alone and bound to oligonucleotides have been solved.

Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB).

The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay.

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment.

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC)-induced DNA damage.

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine.

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol.

Amidst multiple binding orientations on fork DNA, MCM helicase proceeds N-first for unwinding.

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands.

Contacts and context that regulate DNA helicase unwinding and replisome progression.

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex.

Fine-tuning of the replisome: Mcm10 regulates fork progression and regression.

Several decades of research have identified Mcm10 hanging around the replisome making several critical contacts with a number of proteins but with no real disclosed function. Recently, the O'Donnell laboratory has been better able to map the interactions of Mcm10 with a larger Cdc45/GINS/MCM (CMG) unwinding complex placing it at the front of the replication fork. They have shown biochemically that Mcm10 has the impressive ability to strip off single-strand binding protein (RPA) and reanneal complementary DNA strands.

The MCM8/9 complex: A recent recruit to the roster of helicases involved in genome maintenance.

There are several DNA helicases involved in seemingly overlapping aspects of homologous and homoeologous recombination. Mutations of many of these helicases are directly implicated in genetic diseases including cancer, rapid aging, and infertility. MCM8/9 are recent additions to the catalog of helicases involved in recombination, and so far, the evidence is sparse, making assignment of function difficult.

Control of Hexamerization, Assembly, and Excluded Strand Specificity for the Sulfolobus solfataricus MCM Helicase.

A growing body of evidence supports a steric exclusion and wrapping model for DNA unwinding in which hexameric helicases interact with the excluded single-stranded DNA (ssDNA) in addition to the encircled strand. Interactions with the excluded ssDNA have been shown to be mediated primarily by electrostatic interactions, but base stacking with surface-exposed tyrosine residues is an alternative hypothesis. Here, we mutated several external tyrosine and positively charged residues from full-length Sulfolobus solfataricus MCM along the proposed path of excluded strand binding and assessed their impact on DNA unwinding.

Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding.

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal minichromosome maintenance (MCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency.

Coordination and Substitution of DNA Polymerases in Response to Genomic Obstacles.

The ability for DNA polymerases (Pols) to overcome a variety of obstacles in its path to maintain genomic stability during replication is a complex endeavor. It requires the coordination of multiple Pols with differing specificities through molecular control and access to the replisome. Although a number of contacts directly between Pols and accessory proteins have been identified, forming the basis of a variety of holoenzyme complexes, the dynamics of Pol active site substitutions remain uncharacterized.

Characterization of a coupled DNA replication and translesion synthesis polymerase supraholoenzyme from archaea.

The ability of the replisome to seamlessly coordinate both high fidelity and translesion DNA synthesis requires a means to regulate recruitment and binding of enzymes from solution. Co-occupancy of multiple DNA polymerases within the replisome has been observed primarily in bacteria and is regulated by posttranslational modifications in eukaryotes, and both cases are coordinated by the processivity clamp. Because of the heterotrimeric nature of the PCNA clamp in some archaea, there is potential to occupy and regulate specific polymerases at defined subunits.

Mechanistic insights into how CMG helicase facilitates replication past DNA roadblocks.

Before leaving the house, it is a good idea to check for road closures that may affect the morning commute. Otherwise, one may encounter significant delays arriving at the destination. While this is commonly true, motorists may be able to consult a live interactive traffic map and pick an alternate route or detour to avoid being late.