Evidence that DNA polymerase δ proofreads errors made by DNA polymerase α across the Saccharomyces cerevisiae nuclear genome.

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated.

Bursicon regulates wing expansion via PKA in the oriental fruit fly, Bactrocera dorsalis.

Background: Bursicon is a heterodimeric neuropeptide that is involved in many physiological activities such as cuticle tanning, wing expansion, reproduction and immunity in insects. In this study, the role of bursicon in the wing expansion was investigated in Bactrocera dorsalis, an important invasive insect pest in agriculture.

Results: The cDNA sequences and deduced amino acids of bursicon genes (named BdBurs-α and BdBurs-β) were determined, and two proteins typically contained 11 cysteine residues in conserved positions that were highly conserved in other insect species.

Pif1 family helicases promote mutation avoidance during DNA replication.

Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication.

Transcriptome Analysis Reveals Molecular Underpinnings of Common Carp () Under Hypoxia Stress.

As an essential environmental factor that affects the economic benefits of aquaculture, hypoxia is one of the urgent problems to be solved in the aquaculture fish breeding industry. Common carp () is a critical economic fish in China, and at present, there are many breeding strains of common carp with different character advantages in China, including Hebao red carp ( var ) and Songpu mirror carp ( var ). Even if the environmental adaptation of common carp is generally strong, the genetic background of hypoxia tolerance in different strains of common carp is unclear yet.

CYP314A1-dependent 20-hydroxyecdysone biosynthesis is involved in regulating the development of pupal diapause and energy metabolism in the Chinese citrus fruit fly, Bactrocera minax.

Background: Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20-hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear.

How asymmetric DNA replication achieves symmetrical fidelity.

Accurate DNA replication of an undamaged template depends on polymerase selectivity for matched nucleotides, exonucleolytic proofreading of mismatches, and removal of remaining mismatches via DNA mismatch repair (MMR). DNA polymerases (Pols) δ and ε have 3'-5' exonucleases into which mismatches are partitioned for excision in cis (intrinsic proofreading). Here we provide strong evidence that Pol δ can extrinsically proofread mismatches made by itself and those made by Pol ε, independently of both Pol δ's polymerization activity and MMR.

regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis.

Aim: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is the key of the development of diabetic retinopathy (DR), and lncRNA NEAT1 could accelerate EMT in diabetic nephropathy. Meanwhile, as a diabetes susceptibility gene, whether sex-determining region Y-related (SRY) high-mobility group box 4 (SOX4) has relationship with lncRNA NEAT1 in DR remains unclear.

Methods: Firstly, NEAT1, SOX4 and miR-204 were evaluated by qRT-PCR (quantitative reverse-transcriptase PCR) under high glucose condition.

Ribonucleotide incorporation into DNA during DNA replication and its consequences.

Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology.

DNA Polymerase Delta Synthesizes Both Strands during Break-Induced Replication.

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized.

Roles for DNA polymerase δ in initiating and terminating leading strand DNA replication.

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome. Given that ribonucleotide and mismatch incorporation rates by these replicases influence somatic and germline patterns of variation, it is important to understand the details and exceptions to this overall division of labor. Using an improved method to map where these replicases incorporate ribonucleotides during replication, here we present evidence that DNA polymerase δ universally participates in initiating leading strand synthesis and that nascent leading strand synthesis switches from Pol ε to Pol δ during replication termination.

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated.

Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae.

To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication.

Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall.

In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication.

Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2.

Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24.

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method.

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)-associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions.