DNA polymerase zeta can efficiently replicate structures formed by AT/TA repeat sequences and prevent their deletion.

Long AT repeat tracts form non-B DNA structures that stall DNA replication and cause chromosomal breakage. AT repeats are abundant in human common fragile sites (CFSs), genomic regions that undergo breakage under replication stress. Using an in vivo yeast model system containing AT-rich repetitive elements from human CFS FRA16D, we find that DNA polymerase zeta (Pol ζ) is required to prevent breakage and subsequent deletions at hairpin and cruciform forming (AT/TA)n sequences, with little to no role at an (A/T)28 repeat or a control non-structure forming sequence.

Replication of [AT/TA] Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats.

Replication of [AT/TA] microsatellite sequences by human DNA polymerase δ holoenzymes is dependent on dNTP and RPA levels.

Difficult-to-Replicate Sequences (DiToRS) are natural impediments in the human genome that inhibit DNA replication under endogenous replication. Some of the most widely-studied DiToRS are A+T-rich, high "flexibility regions," including long stretches of perfect [AT/TA] microsatellite repeats that have the potential to collapse into hairpin structures when in single-stranded DNA (ssDNA) form and are sites of recurrent structural variation and double-stranded DNA (dsDNA) breaks. Currently, it is unclear how these flexibility regions impact DNA replication, greatly limiting our fundamental understanding of human genome stability.

Accurate sequencing of DNA motifs able to form alternative (non-B) structures.

Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases.

Special Issue "DNA Replication/Repair, and the DNA Damage Response in Human Disease".

Mutations of numerous genes involved in DNA replication, DNA repair, and DNA damage response (DDR) pathways lead to a variety of human diseases, including aging and cancer [...

Nontraditional Roles of DNA Polymerase Eta Support Genome Duplication and Stability.

DNA polymerase eta (Pol η) is a Y-family polymerase and the product of the gene. Autosomal recessive inheritance of mutations is the cause of the xeroderma pigmentosum variant, a cancer predisposition syndrome. This review summarizes mounting evidence for expanded Pol η cellular functions in addition to DNA lesion bypass that are critical for maintaining genome stability.

Variation in G-quadruplex sequence and topology differentially impacts human DNA polymerase fidelity.

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown.

Impact of G-Quadruplexes and Chronic Inflammation on Genome Instability: Additive Effects during Carcinogenesis.

Genome instability is an enabling characteristic of cancer, essential for cancer cell evolution. Hotspots of genome instability, from small-scale point mutations to large-scale structural variants, are associated with sequences that potentially form non-B DNA structures. G-quadruplex (G4) forming motifs are enriched at structural variant endpoints in cancer genomes.

Selection and thermostability suggest G-quadruplexes are novel functional elements of the human genome.

Approximately 1% of the human genome has the ability to fold into G-quadruplexes (G4s)-noncanonical strand-specific DNA structures forming at G-rich motifs. G4s regulate several key cellular processes (e.g.

Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion.

DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases η and κ. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation.

Non-B DNA: a major contributor to small- and large-scale variation in nucleotide substitution frequencies across the genome.

Approximately 13% of the human genome can fold into non-canonical (non-B) DNA structures (e.g. G-quadruplexes, Z-DNA, etc.

Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D.

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication.

Detours to Replication: Functions of Specialized DNA Polymerases during Oncogene-induced Replication Stress.

Incomplete and low-fidelity genome duplication contribute to genomic instability and cancer development. Difficult-to-Replicate Sequences, or DiToRS, are natural impediments in the genome that require specialized DNA polymerases and repair pathways to complete and maintain faithful DNA synthesis. DiToRS include non B-DNA secondary structures formed by repetitive sequences, for example within chromosomal fragile sites and telomeres, which inhibit DNA replication under endogenous stress conditions.

DNA Polymerase Eta Prevents Tumor Cell-Cycle Arrest and Cell Death during Recovery from Replication Stress.

: Neoplastic transformation and genome instability are enhanced by replication stress, conditions that slow or stall DNA replication forks. Consequently, cancer cells require multiple enzymes and checkpoint signaling pathways to mitigate replication stress for their viability and proliferation. Targeting proteins that enhance cancer cell survival during replication stress is a recent approach in clinical strategies, especially when targets produce synthetic lethality.

Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL).

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress.

Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats.

Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to nongenetically encoded transcript variants, or RNA-DNA differences (RDDs).

Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites.

Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites.

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins.

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo.

Microsatellite interruptions stabilize primate genomes and exist as population-specific single nucleotide polymorphisms within individual human genomes.

Interruptions of microsatellite sequences impact genome evolution and can alter disease manifestation. However, human polymorphism levels at interrupted microsatellites (iMSs) are not known at a genome-wide scale, and the pathways for gaining interruptions are poorly understood. Using the 1000 Genomes Phase-1 variant call set, we interrogated mono-, di-, tri-, and tetranucleotide repeats up to 10 units in length.

Interplay between DNA repair and inflammation, and the link to cancer.

DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer.

DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis.

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role.

Mature microsatellites: mechanisms underlying dinucleotide microsatellite mutational biases in human cells.

Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases.