Components Of DNA Replication Research Articles

Crotonylation of MCM6 enhances chemotherapeutics sensitivity of breast cancer via inducing DNA replication stress.

Breast cancer is associated with high morbidity and mortality, which are closely influenced by protein post-translational modifications (PTMs). Lysine crotonylation (Kcr) serves as a newly identified PTM type that plays a role in various biological processes; however, its involvement in breast cancer progression remains unclear. Minichromosome maintenance 6 (MCM6) is a critical component of DNA replication and has been previous confirmed to exhibit a significant role in tumorigenesis. Despite this, a comprehensive analysis of MCM6, particularly regarding its modifications in breast cancer is lacking. In this study, we found MCM6 is upregulated in breast invasive carcinoma (BRCA) and is associated with poorer overall survival by regulating the DNA damage repair mechanisms. Furthermore, MCM6-knockdown resulted in decreased cell proliferation and inhibited the DNA replication, leading to DNA replication stress and sustained DNA damage, thereby enhancing the chemotherapeutic sensitivity of breast cancer. Additionally, SIRT7-mediated crotonylation of MCM6 at K599 (MCM6-K599cr) was significantly upregulated in response to DNA replication stress, primarily due to the disassemebly of the MCM2-7 complex and regulated by RNF8-mediated ubiquitination. Concurrently, kaempferol, which acts as a regulator of SIRT7, was found to enhance the Kcr level of MCM6, reducing tumour weight, particular when combined with paclitaxel, highlighting its potential chemotherapeutic target for BRCA therapy.

Comparative Analysis of 1,9-Dimethyl-Methylene Blue and Toluidine Blue Interactions with ct-DNA, G-Quadruplex DNA, and ssDNA

This study presents a comprehensive investigation into the interactions of two distinct synthetic dyes—1,9-Dimethyl-Methylene Blue and Toluidine Blue —with different DNA structures, namely calf thymus DNA (ct-DNA), G-quadruplex DNA, and single stranded DNA (ssDNA). The objective of this research is to elucidate the molecular affinities of these dyes for specific DNA structures and explore their potential applications in molecular biology and diagnostics. The experimental approach involved detailed UV-visible spectroscopic analyses, to probe the binding affinities of each dye with ct-DNA, G-quadruplex DNA, and ssDNA. The study aimed to assess the selectivity of these dyes towards the unique structural features of each DNA entity. The binding stoichiometry is defined from Job’s method. The selectivity of the dyes towards DNA also investigated with competitive dialysis experiments. The binding stoichiometry were 1:1 for 1,9-Dimethyl-Methylene Blue and Toluidine Blue and G-quadruplex DNA or ssDNA. Besides, results indicate that 1,9-Dimethyl-Methylene Blue and Toluidine Blue exhibit a pronounced affinity for G-quadruplex DNA, and ct-DNA. While single-stranded DNA is a fundamental component of DNA replication and transcription, our dyes exhibit lower affinity for this structure. The selectivity is advantageous, as it allows for the discrimination between single-stranded and structured DNA regions. The potential applications in studying DNA dynamics and unwinding processes are vast.

Comparative Analysis of Cluster of Differentiation 57 and Proliferating Cell Nuclear Antigen Expression in Different Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The immune defense against tumor cells is mainly mediated by the natural killer (NK) cells. Cluster of differentiation 57 (CD57) is a 110-kd glycoprotein, typically expressed by the NK cells, attacks the cancer cells and inhibits tumor development. Proliferating cell nuclear antigen (PCNA) is a 36-kd auxiliary protein for DNA polymerase delta that correlates with cell proliferation and DNA synthesis. It is an essential component of DNA replication, DNA recombination, and DNA repair. The uncoordinated proliferation of PCNA protein characterizes the biological behavior of malignant lesions. The aim of the present study is to compare and correlate the expression of CD57 and PCNAin different grades of oral squamous cell carcinoma (OSCC) by immunohistochemistry. This retrospective analysis comprises 30 samples of various grades of OSCCsand10 samples of healthy mucosa.Sections of 4-5 µm thickness were done and stained with monoclonal anti-PCNA and anti-CD57 antibodies.The statistical package for social science (SPSS) version 16.0 software (IBM Corp., Armonk, NY) was used to analyze the data in this study. The expression of CD57 and PCNA was compared and correlated between the groups using analysis of variance (ANOVA) post hoc, Dunnet t-test, and Pearson's correlation coefficient test.For statistical significance, a p-value of 0.05 or less was used. A significant decrease in CD57 labeling index was seen from well-differentiated squamous cell carcinoma (16.63 ± 2.33) to poorly differentiated squamous cell carcinoma (5.53 ± 1.20) whereas the significant increase in PCNA labeling index was noted from well-differentiatedsquamous cell carcinoma (45.88 ± 2.20), followed by moderately differentiated and poorly differentiated squamous cell carcinoma (72.77 ± 4.35). The combination of CD57 and PCNA biomarkers appears to be good indicators of the immune status of the patient and the aggressiveness of the lesion.

A Useful Tool for the Identification of DNA-Binding Proteins Using Graph Convolutional Network

Background: DNA and protein are important components of living organisms. DNA binding protein is a helicase, which is a protein specifically responsible for binding to DNA single- stranded regions. It is a necessary component for DNA replication, recombination and repair, and plays a key role in the function of various biomolecules. Although there are already some classification prediction methods for this protein, the use of graph neural networks for this work is still limited. Objective: The classification of unknown protein sequences into the correct categories, subcategories and families is important for biological sciences. In this article, using graph neural networks, we developed a novel predictor GCN-DBP for protein classification prediction. Methods: Each protein sequence is treated as a document in this study, and then segment the words according to the concept of k-mer, thereby, finally achieving the purpose of segmenting the document. This research aims to use document word relationships and word co-occurrence as a corpus to construct a text graph, and then learn protein sequence information by two-layer graph convolutional networks. Results: Finally, we tested GCN-DBP on the independent data set PDB2272, and its accuracy reached 64.17% and MCC was 28.32%. Moreover, in order to compare the proposed method with other existing methods, we have conducted more experiments. Conclusion: The results show that the proposed method is superior to the other four methods and will be a useful tool.

An Agricultural Perspective for Combating Antibiotic Resistance: A Literature Review

Introduction: The overuse of antibiotics has led to a surge of antibiotic resistant bacteria in recent decades. Animal agriculture has proven to be a significant contributor to this overuse. The investigation of potential alternatives to antibiotics in animal agriculture is thus warranted. Methods: We conducted a literature review exploring four alternatives for antibiotic use in an animal agricultural setting: phytochemicals, antimicrobial peptides, probiotics, and bacteriophages. Results: Four different types of antibiotic alternatives were evaluated. The first one is phytochemicals, a broad group consisting of five subtypes: alkaloids, carotenoids, polyphenols, terpenoids, and organosulfur compounds. Many of these display antibacterial properties such as interacting with the cytoplasmic membrane, immunomodulatory activities, inhibiting specific components of DNA replication, and even inactivating specific enzymes vital for the survival of bacteria. All these subtypes displayed various antibacterial properties in animal studies surrounding poultry and livestock. The second alternative is antimicrobial peptides, which have shown to be effective in treating conditions such as post-weaning stress and necrotic enteritis in various animals. The third alternative is probiotics, which have demonstrated both growth promotion and disease prevention properties. One study also concluded that probiotics provide financial benefits to farmers. The fourth antibiotic alternative is bacteriophages, a class of viruses that infect bacteria. Phages have shown disease prevention and growth promotion properties; they also can prevent the transmission of zoonotic diseases. Discussion: Phytochemicals, antimicrobial peptides, probiotics, and bacteriophages all demonstrate the capability of acting as antibiotic alternatives. Each of these alternatives are unique with their own advantages and disadvantages; hence, the applicability is broad. Conclusion: Four antibiotic alternatives (phytochemicals, antimicrobial peptides, probiotics, and bacteriophages) were researched for disease prevention and growth promotion properties, inherent functions of antibiotics. The applicability of such options in livestock and poultry is vast. Therefore, given more funding for research studies and policy changes, many of these options can be implemented if not already.

Elevated expression of minichromosome maintenance 3 indicates poor outcomes and promotes G1/S cell cycle progression, proliferation, migration and invasion in colorectal cancer.

Background: The minichromosome maintenance (MCM) family, a core component of DNA replication, is involved in cell cycle process. Abnormal proliferation has been identified as a crucial process in the evolution of colorectal cancer (CRC). However, the roles of the MCM family in CRC remain largely unknown.Methods: Here, the expression, prognostic significance and functions of the MCM family in CRC were systematically analyzed through a series of online databases including CCLE, Oncomine, HPA, cBioPortal and cancerSEA.Results: We found all MCM family members were highly expressed in CRC, but only elevation of MCM3 expression was associated with poor prognosis of patients with CRC. Further in vitro and in vivo experiments were performed to examine the role of MCM3 in CRC. Analysis of CCLE database and qRT-PCR assay confirmed that MCM3 was overexpressed in CRC cell lines. Moreover, knockdown of MCM3 significantly suppressed transition of G1 to S phase in CRC cells. Furthermore, down-regulation of MCM3 inhibited CRC cell proliferation, migration, invasion and promoted apoptosis.Conclusion: These findings reveal that MCM3 may function as an oncogene and a potential prognosis biomarker. Thus, the association between abnormal expression of MCM3 and the initiation of CRC deserves further exploration.

Distinct expression and prognostic values of the replication protein A family in gastric cancer.

The replication protein A (RPA)1-4 family are single-stranded DNA-binding proteins that are essential components of DNA replication, repair and recombination, and cell cycle regulation. The present study aimed to evaluate the prognostic value of the RPA family members in patients with gastric cancer (GC), using datasets retrieved from the Oncomine public database. Datasets were retrieved for the purpose of comparing the RPA expression levels between GC and normal tissues. Additionally, Kaplan-Meier analysis was used to compare the overall survival (OS) times of GC patients that expressed different levels of RPA proteins. RPA1, 2, and 3 expression levels were all significantly upregulated in gastric intestinal-type, diffuse gastric, and gastric mixed adenocarcinomas, compared with those in normal mucosal tissues. Moreover, high mRNA expression levels of RPA3 and 4 predicted poorer OS times in all GCs, as well as patients with human epidermal growth factor receptor 2-negative and -positive GC. The high-risk group, separated by RPA signature, showed a poorer outcome than the low-risk group. RPA3 was the most strongly correlated with CD4+ T-cell levels. In conclusion, RPAs are novel prognostic indicators in GC, and can also predict the features of immunological diseases. Future experimental investigation into the roles of RPAs concerning the pathogenesis and development of GC may provide a novel biomarker or therapeutic target, improving the prognosis of patients with GC.

Structure-Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System.

Plants are sessile organisms, and their DNA is particularly exposed to damaging agents. The integrity of plant mitochondrial and plastid genomes is necessary for cell survival. During evolution, plants have evolved mechanisms to replicate their mitochondrial genomes while minimizing the effects of DNA damaging agents. The recombinogenic character of plant mitochondrial DNA, absence of defined origins of replication, and its linear structure suggest that mitochondrial DNA replication is achieved by a recombination-dependent replication mechanism. Here, I review the mitochondrial proteins possibly involved in mitochondrial DNA replication from a structural point of view. A revision of these proteins supports the idea that mitochondrial DNA replication could be replicated by several processes. The analysis indicates that DNA replication in plant mitochondria could be achieved by a recombination-dependent replication mechanism, but also by a replisome in which primers are synthesized by three different enzymes: Mitochondrial RNA polymerase, Primase-Helicase, and Primase-Polymerase. The recombination-dependent replication model and primers synthesized by the Primase-Polymerase may be responsible for the presence of genomic rearrangements in plant mitochondria.

Centromere Stability: The Replication Connection.

The fission yeast centromere, which is similar to metazoan centromeres, contains highly repetitive pericentromere sequences that are assembled into heterochromatin. This is required for the recruitment of cohesin and proper chromosome segregation. Surprisingly, the pericentromere replicates early in the S phase. Loss of heterochromatin causes this domain to become very sensitive to replication fork defects, leading to gross chromosome rearrangements. This review examines the interplay between components of DNA replication, heterochromatin assembly, and cohesin dynamics that ensures maintenance of genome stability and proper chromosome segregation.

PC-1 works in conjunction with E3 ligase CHIP to regulate androgen receptor stability and activity

The androgen receptor (AR) is not only a ligand-dependent transcription factor, but also functions as a licensing factor, a component of DNA replication, which is degraded during mitosis. Furthermore, the deregulation of AR activity is involved in the initiation of prostate cancer and contributes to castration resistant prostate cancer (CRPC). While AR degradation is known to occur primarily through a proteasome-mediated pathway, very little is known about how this process is regulated, especially in M phase. PC-1 is an androgen-responsive factor and expresses specificity in prostate cancer, with higher expression noted at G2/M. In this study, PC-1 was shown to interact with AR and E3 ligase CHIP (Carboxy-terminus of Hsc70 Interacting Protein) and to enhance AR/CHIP interactions, thereby decreasing AR stability. Moreover, PC-1 was found to act in conjunction with CHIP in the decreasing of AR via ubiquitination, with the subsequent degradation predominantly occurring during M phase. PC-1 was also found to repress AR transcriptional activity in androgen-dependent and androgen-independent prostate cancer cells and attenuate the growth inhibition of AR. In conclusion, these findings should provide new clues regarding the modulation of AR turnover and activity via PC-1 and reveals an essential role of PC-1 in AR signaling.

DNA-Directed Polymerase Subunits Play a Vital Role in Human Telomeric Overhang Processing.

Telomeres consist of TTAGGG repeats bound by the shelterin complex and end with a 3' overhang. In humans, telomeres shorten at each cell division, unless telomerase (TERT) is expressed and able to add telomeric repeats. For effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. Recent studies have discovered a link between different activities necessary to process telomeres in the S phase of the cell cycle to reform a proper overhang. Notably, the human CST complex (CTC1/STN1/TEN1), known to interact functionally with the polymerase complex (POLA/primase), was shown to be important for telomere processing. Here, focus was paid to the catalytic (POLA1/p180) and accessory (POLA2/p68) subunits of the polymerase, and their mechanistic roles at telomeres. We were able to detect p68 and p180 at telomeres in S-phase using chromatin immunoprecipitation. We could also show that the CST, shelterin, and polymerase complexes interact, revealing contacts occurring at telomeres. We found that the polymerase complex could associate with telomerase activity. Finally, depletion of p180 by siRNA led to increased overhang amounts at telomeres. These data support a model in which the polymerase complex is important for proper telomeric overhang processing through fill-in synthesis, during S phase. These results shed light on important events necessary for efficient telomere maintenance and protection. This study describes the interplay between DNA replication components with proteins that associate with chromosome ends, and telomerase. These interactions are proposed to be important for the processing and protection of chromosome ends.

Highlights of This Issue

High-grade serous ovarian cancer (HGSOC) remains one of the most lethal cancer types despite aggressive standard treatment. HGSOC is characterized by widespread recurrent regions of copy number gain and loss. Luo and colleagues performed genomic analyses and interrogated a diverse number of genes that are recurrently amplified in HGSOC and are essential for cancer proliferation and survival in ovarian cancer cell lines. One of the genes, FGF receptor substrate 2 (FRS2), encodes an adaptor protein and induces transformation in immortalized human cell lines. These results implicate FRS2 as an oncogene and a potential therapeutic target in a subset of HGSOC that harbors FRS2 amplifications.Telomere maintenance is essential for oncogenic transformation of human cells through the bypass of senescence, known to be an important tumor-suppressive mechanism. In addition to the three activities known to regulate telomere function (telomerase, shelterin, and the CST complex), a fourth complex was found to interact with these, the POLA/primase complex. Diotti and colleagues demonstrate that the polymerase-α (p180) enzyme and its regulatory subunit (p68) interact with telomeric complexes and are important for telomere function. Therefore specific DNA replication components play a role at telomeres and can be seen as relevant targets against tumor cell growth.Neuroblastoma is a common solid tumor in children, and high-risk patients often present with genomic alterations suggesting the importance of DNA repair fidelity. Therefore, it is critical to identify components of the alternative NHEJ pathway as promising new therapeutic targets. Newman and colleagues identify a potential mechanistic explanation for the frequent chromosomal rearrangements found in neuroblastoma tumors. Importantly, clinical tumor specimens with high mRNA expression of alternative NHEJ pathway components have worse survival outcomes than those with low expression. These findings are of interest to both basic scientists in the DNA repair community and to clinical oncologists.HIF1α is a master regulator of adaptive responses to hypoxia, and its activation leads to metabolic reprogramming, angiogenesis, and tumor progression. Its role in hypoxic signaling in cancer stem cells (CSC) is not well known. Marhold and colleagues show that elevated HIF1α in a prostate CSC subpopulation leads to deregulated PI3K/AKT/mTOR. This suggests that CSCs rely on HIF1α-mediated inhibition of mTOR, which promotes CSC quiescence and survival in the hypoxic tumor microenvironment. Downregulation of HIF1α consequently attenuates this effect and increases proliferation of CSCs. This study also implicates primary resistance of CSCs to selective mTOR inhibition and provides a rationale for the use of dual PI3K/mTOR inhibitors.

DNA replication components as regulators of epigenetic inheritance—lesson from fission yeast centromere

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.

Role of DNA damage response pathways in preventing carcinogenesis caused by intrinsic replication stress

Defective DNA replication can result in genomic instability, cancer, and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4Chaos3/Chaos3 (“Chaos3”) mouse model which, by virtue of an amino acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ATM double strand break response pathway (Atm, p21/Cdkn1a, Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. ATM deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development.

PCNA tyrosine phosphorylation by EGFR promotes genome instability

The proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and repair, including DNA mismatch repair (MMR), an essential mechanism that ensures replication fidelity. PCNA is required for MMR at both the initiation and re‐synthesis steps. Recent studies have shown that PCNA is phosphorylated at tyrosine 211 (Y211) by epidermal growth factor receptor (EGFR), whose overexpression is associated with during tumor progression. We therefore hypothesize that the EGFR tumor‐promotion function is through PCNA Y211 phosphorylation by altering the MMR function. To test this hypothesis, we directly examined the influence of PCNA Y211 phosphorylation on MMR activity using a functional in vitro repair assay. We demonstrate here that nuclear extracts derived from tumor cells with high levels of phosphorylated PCNA are defective in MMR, and that the deficiency can be restored by purified non‐phosphorylated PCNA, suggesting that PCNA Y211 phosphorylation inhibits MMR. The inhibition was due to the inability of the phosphorylated PCNA to interact with mismatch recognition proteins MutSα and MutSβ. We also show that the PCNA‐phosphorylated nuclear extracts have a delayed DNA resynthesis during MMR, which is coupled with nucleotide misincorporations, leading to an elevated mutation frequency. Our study therefore demonstrates that a posttranslational modification promotes genome instability and tumor progression by negatively regulating the MMR function and DNA synthesis. Thus, the work provides a potential new biomarker for cancer progression.

Small molecule inhibitors of PCNA/PIP-box interaction suppress translesion DNA synthesis

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5μM inhibited cellular DNA replication at 10μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.

DnaN clamp zones provide a platform for spatiotemporal coupling of mismatch detection to DNA replication

Mismatch repair (MMR) increases the fidelity of DNA replication by identifying and correcting replication errors. Processivity clamps are vital components of DNA replication and MMR, yet the mechanism and extent to which they participate in MMR remains unclear. We investigated the role of the Bacillus subtilis processivity clamp DnaN, and found that it serves as a platform for mismatch detection and coupling of repair to DNA replication. By visualizing functional MutS fluorescent fusions in vivo, we find that MutS forms foci independent of mismatch detection at sites of replication (i.e. the replisome). These MutS foci are directed to the replisome by DnaN clamp zones that aid mismatch detection by targeting the search to nascent DNA. Following mismatch detection, MutS disengages from the replisome, facilitating repair. We tested the functional importance of DnaN-mediated mismatch detection for MMR, and found that it accounts for 90% of repair. This high dependence on DnaN can be bypassed by increasing MutS concentration within the cell, indicating a secondary mode of detection in vivo whereby MutS directly finds mismatches without associating with the replisome. Overall, our results provide new insight into the mechanism by which DnaN couples mismatch recognition to DNA replication in living cells.

Spartan/C1orf124, a Reader of PCNA Ubiquitylation and a Regulator of UV-Induced DNA Damage Response

PCNA is a key component of DNA replication and repair machineries. DNA damage-induced PCNA ubiquitylation serves as a molecular mark to orchestrate postreplication repair. Here, we have identified and characterized Spartan, a protein that specifically recognizes ubiquitylated PCNA and plays an important role in cellular resistance to UV radiation. In vitro, Spartan engages ubiquitylated PCNA via both a PIP box and a UBZ domain. In cells, Spartan is recruited to sites of UV damage in a manner dependent upon the PIP box, the UBZ domain, and PCNA ubiquitylation. Furthermore, Spartan colocalizes and interacts with Rad18, the E3 ubiquitin ligase that modifies PCNA. Surprisingly, while Spartan is recruited by ubiquitylated PCNA, knockdown of Spartan compromised chromatin association of Rad18, monoubiquitylation of PCNA, and localization of Pol η to UV damage. Thus, as a "reader" of ubiquitylated PCNA, Spartan promotes an unexpected feed-forward loop to enhance PCNA ubiquitylation and translesion DNA synthesis.

Bypass of a Nick by the Replisome of Bacteriophage T7

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.

Strand Annealing and Terminal Transferase Activities of a B-family DNA Polymerase

DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.