Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.

DNA Breaks and Gaps Target Retroviral Integration

Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the virus-encoded integrase (IN) and the reverse transcribed (RT) virus copy DNA (cDNA). Previous studies suggested that integration was limited by intasome-host DNA recognition progressions. Using single molecule Forster resonance energy transfer (smFRET) we show that PFV intasomes pause at nicked and gapped DNA, which targeted site-directed integration without inducing significant intasome conformational alterations. Base excision repair (BER) components that affect retroviral integration in vivo produce similar nick/gap intermediates during DNA lesion processing. Intasome pause dynamics was modified by the 5′-nick-gap chemistry, while an 8-oxo-guanine lesion, a mismatch, or a nucleotide insertion that induce backbone flexibility and/or static bends had no effect. These results suggest that dynamic often non-productive intasome-DNA interactions may be modulated to target retroviral integration.

A Novel ATM Gene Mutation Affecting Splicing in an Ataxia-Telangiectasia Patient

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by progressive ataxia, choreoathetosis and immunodeficiency beginning in early childhood. An 8-year-old girl was referred with a diagnosis of AT. She had gait disturbance and dysarthria for 3years. Multiple cutaneous telangiectases were observed on her face, trunk and limbs. Sequence analysis of the <i>ATM</i> gene revealed a homozygous c.7308–15A&#x3e;G mutation in IVS49. Human Splicing Finder predicted that the mutation could activate an intronic cryptic acceptor site. We designed primers for amplification of related exons (48–50) from cDNA for evaluating splicing pattern. Sequencing of <i>ATM</i> exons 48–50 revealed a 14-nucleotide insertion from intron 49, between exons 49 and 50, resulting in premature termination of translation at codon 2439. To conclude, we report a novel mutation in a classical AT case, which resulted in an alternatively spliced transcript and was predicted to form a truncated protein or null protein due to nonsense-mediated decay.

Waardenburg syndrome type 2A in a large Iranian family with a novel MITF gene mutation

Background The characteristics of Waardenburg syndrome (WS) as a scarce heritable disorder are sensorineural hearing loss and deficits of pigmentation in the skin, hair, and eye. Here, clinical features and detection of the mutation in the MITF gene of WS2 patients are reported in a sizable Iranian family. Methods A man aged 28-years represented with symptoms of mild unilateral hearing loss (right ear), complete heterochromia iridis, premature graying prior to 30 years of age, and synophrys. In this research, there was a sizable family in Iran comprising three generations with seven WS patients and two healthy members. Whole exome sequencing was applied for proband for the identification of the candidate genetic mutations associated with the disease. The detected mutation in proband and investigated family members was validated by PCR-Sanger sequencing. Results A novel heterozygous mutation, NM_198159.3:c.1026dup p.(Asn343Glufs*27), in exon 9 of the MITF gene co-segregated with WS2 in the affected family members. The variant was forecasted as a disease-causing variant by the Mutation Taster. According to the UniProt database, this variant has been located in basic helix-loop-helix (bHLH) domain of the protein with critical role in DNA binding. Conclusions A frameshift was caused by a nucleotide insertion, c.1026dup, in exon 9 of the MITF gene. This mutation is able to induce an early termination, resulting in forming a truncated protein capable of affecting the normal function of the MITF protein. Helpful information is provided through an exactly described mutations involved in WS to clarify the molecular cause of clinical characteristics of WS and have a contribution to better genetic counseling of WS patients.

A well conserved archaeal B-family polymerase functions as a mismatch and lesion extender

ABSTRACTB-family DNA polymerases (PolBs) of different groups are widespread in Archaea and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that are poorly characterized is PolB2 whose members occur in many archaea but are predicted as an inactivated form of DNA polymerase. Herein, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase harbors a robust nucleotide incorporation activity, but devoid of the 3’-5’ exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. Together, these data suggested Dpo2 functions as a mismatch and lesion extender, representing a novel type of PolB that is primarily involved in DNA damage repair in Archaea. Insights were also gained into the functional adaptation of the motif C in the mismatch extension of the B-family DNA polymerases.

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Characterization and complete genome sequence of bacteriophage vB_Vc_SrVc2, a marine phage that infects Vibrio campbellii

Vibrio campbellii is widely distributed in the marine environment and is an important pathogen of aquatic organisms such as shrimp, fish, and mollusks. The emergence of multi-drug resistance among these bacteria resulted in a worldwide public health problem, which requires alternative treatment approaches such as phage therapy. In the present study, we isolated a phage vB_Vc_SrVc2 from white shrimp hepatopancreas with symptoms of AHPND. Phage vB_Vc_SrVc2 is a member of the genus Maculvirus and the family Autographiviridae, with high lytic ability against Vibrio isolates. Phage has a high resistance to a broad range of temperatures, salinity, UV radiation and chloroform. The genome size was 43,157 bp, with a GC content of 49.2% that encodes 49 putative ORFs, no tRNAs, showed three single nucleotide polymorphisms, two small deletions and one nucleotide insertion compared to SrVc9, showing slightly different infectivity profiles.. No lysogeny related genes were detected in vB_Vc_SrVc2 genome. Overall phage vB_Vc_SrVc2 has a good potential for therapeutic use in the aquaculture industry against V. campbellii infections.

Three Novel Mutations in CYB5R3 Gene Causing NADH-cytochrome b5 Reductase Enzyme Deficiency Leads to Recessive Congenital Methaemoglobinemia

Two types of recessive congenital methaemoglobinemia (RCM) is caused by NADH-dependent cytochrome b5 reductase enzyme deficiency encoded by CYB5R3 gene. RCM-I is characterized by higher methaemoglobin levels (>2 g/dL), causing only cyanosis, whereas RCMR-II is associated with cyanosis with neurological impairment. The present study discovered three novel homozygous pathogenic variants of CYB5R3 causing RCM I and II in four unrelated Indian patients. In patient-1 and patient-2 of are of RCM type I caused due to novel c.175C>T (p.Arg59Cys) and other reported c.469T>C (p.Phe157Ser) missense pathogenic variants respectively, whereas patient-3 and patient-4 presented with the RCM type II are related to developmental delay with cyanosis since birth due to a novel homozygous (g.25679_25679delA) splice-site deletion and novel homozygous c.824_825insC (p.Pro278ThrfsTer367) single nucleotide insertion. The CYB5R3 transcript levels were estimated by qRT-PCR in the splice-site deletion, which was 0.33fold of normal healthy control. The insertion of nucleotide C resulted in a frame-shift of termination codon are associated with neurological impairment. This study can help to conduct genetic counselling and, subsequently, prenatal diagnosis of high-risk genetic disorders.

Analysis of B-cell receptor repertoires in COVID-19 patients using deep embedded representations of protein sequences

Analyzing B-cell receptor (BCR) repertoires is immensely useful in evaluating one's immunological status. Conventionally,repertoire analysis methods have focused on comprehensive assessment of clonal compositions, including V(D)J segment usage, nucleotide insertion/deletion, and amino acid distribution. Here, we introduce a novel computational approach that applies deep-learning based protein embedding techniques to analyze BCR repertoires. By selecting the most frequently occurring BCR sequences in a given repertoire and computing the sum of the vector representations of these sequences, we represent an entire repertoire as a 100-dimensional vector and eventually as a single data point in vector space. We demonstrate that our new approach enables us to not only accurately cluster repertoires of COVID-19 patients and healthy subjects, but also efficiently track minute changes in immunity conditions as patients undergo a course of treatment over time. Furthermore, using the distributed representations, we successfully trained an XGBoost classification model that achieved over 87% mean accuracy rate given a repertoire of CDR3 sequences.