bims-midmar 2022-01-30 papers

Genome Res. 2022 Jan 24. pii: gr.276013.121. [Epub ahead of print]

Mitochondrial DNA variation across 56,434 individuals in gnomAD.

Genome Aggregation Database Consortium

Genomic databases of allele frequency are extremely helpful for evaluating clinical variants of unknown significance; however, until now, databases such as the Genome Aggregation Database (gnomAD) have focused on nuclear DNA and have ignored the mitochondrial genome (mtDNA). Here we present a pipeline to call mtDNA variants that addresses three technical challenges: (i) detecting homoplasmic and heteroplasmic variants, present respectively in all or a fraction of mtDNA molecules, (ii) circular mtDNA genome, and (iii) misalignment of nuclear sequences of mitochondrial origin (NUMTs). We observed that mtDNA copy number per cell varied across gnomAD cohorts and influenced the fraction of NUMT-derived false-positive variant calls, which can account for the majority of putative heteroplasmies. To avoid false positives, we excluded contaminated samples, cell lines, and samples prone to NUMT misalignment due to few mtDNA copies. Furthermore, we report variants with heteroplasmy greater than 10%. We applied this pipeline to 56,434 whole genome sequences in the gnomAD v3.1 database that includes individuals of European (58%), African (25%), Latino (10%), and Asian (5%) ancestry. Our gnomAD v3.1 release contains population frequencies for 10,850 unique mtDNA variants at more than half of all mtDNA bases. We report frequencies within each nuclear ancestral population and mitochondrial haplogroup. Homoplasmic variants account for most variant calls (98%) and unique variants (85%). We observed that 1/250 individuals carry a pathogenic mtDNA variant with heteroplasmy above 10%. These mtDNA population allele frequencies are freely accessible and will aid in diagnostic interpretation and research studies.

DOI: https://doi.org/10.1101/gr.276013.121

EBioMedicine. 2022 Jan 24. pii: S2352-3964(22)00004-4. [Epub ahead of print]76 103815

Comprehensive summary of mitochondrial DNA alterations in the postmortem human brain: A systematic review.

Alba Valiente-Pallejà, Juan Tortajada, Bengisu K Bulduk, Elisabet Vilella, Glòria Garrabou, Gerard Muntané, Lourdes Martorell.

BACKGROUND: Mitochondrial DNA (mtDNA) encodes 37 genes necessary for synthesizing 13 essential subunits of the oxidative phosphorylation system. mtDNA alterations are known to cause mitochondrial disease (MitD), a clinically heterogeneous group of disorders that often present with neuropsychiatric symptoms. Understanding the nature and frequency of mtDNA alterations in health and disease could be a cornerstone in disentangling the relationship between biochemical findings and clinical symptoms of brain disorders. This systematic review aimed to summarize the mtDNA alterations in human brain tissue reported to date that have implications for further research on the pathophysiological significance of mtDNA alterations in brain functioning.
METHODS: We searched the PubMed and Embase databases using distinct terms related to postmortem human brain and mtDNA up to June 10, 2021. Reports were eligible if they were empirical studies analysing mtDNA in postmortem human brains.
FINDINGS: A total of 158 of 637 studies fulfilled the inclusion criteria and were clustered into the following groups: MitD (48 entries), neurological diseases (NeuD, 55 entries), psychiatric diseases (PsyD, 15 entries), a miscellaneous group with controls and other clinical diseases (5 entries), ageing (20 entries), and technical issues (5 entries). Ten entries were ascribed to more than one group. Pathogenic single nucleotide variants (pSNVs), both homo- or heteroplasmic variants, have been widely reported in MitD, with heteroplasmy levels varying among brain regions; however, pSNVs are rarer in NeuD, PsyD and ageing. A lower mtDNA copy number (CN) in disease was described in most, but not all, of the identified studies. mtDNA deletions were identified in individuals in the four clinical categories and ageing. Notably, brain samples showed significantly more mtDNA deletions and at higher heteroplasmy percentages than blood samples, and several of the deletions present in the brain were not detected in the blood. Finally, mtDNA heteroplasmy, mtDNA CN and the deletion levels varied depending on the brain region studied.
INTERPRETATION: mtDNA alterations are well known to affect human tissues, including the brain. In general, we found that studies of MitD, NeuD, PsyD, and ageing were highly variable in terms of the type of disease or ageing process investigated, number of screened individuals, studied brain regions and technology used. In NeuD and PsyD, no particular type of mtDNA alteration could be unequivocally assigned to any specific disease or diagnostic group. However, the presence of mtDNA deletions and mtDNA CN variation imply a role for mtDNA in NeuD and PsyD. Heteroplasmy levels and threshold effects, affected brain regions, and mitotic segregation patterns of mtDNA alterations may be involved in the complex inheritance of NeuD and PsyD and in the ageing process. Therefore, more information is needed regarding the type of mtDNA alteration, the affected brain regions, the heteroplasmy levels, and their relationship with clinical phenotypes and the ageing process.
FUNDING: Hospital Universitari Institut Pere Mata; Institut d'Investigació Sanitària Pere Virgili; Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (PI18/00514).

Keywords: Ageing; Mitochondrial DNA; Mitochondrial diseases; Neurological diseases; Postmortem; Psychiatric diseases

DOI: https://doi.org/10.1016/j.ebiom.2022.103815

J Biochem. 2022 Jan 26. pii: mvac005. [Epub ahead of print]

Into the matrix: current methods for mitochondrial translation studies.

Antonios Apostolopoulos, Shintaro Iwasaki.

In addition to the cytoplasmic translation system, eukaryotic cells house additional protein synthesis machinery in mitochondria. The importance of this in organello translation is exemplified by clinical pathologies associated with mutations in mitochondrial translation factors. Although a detailed understanding of mitochondrial translation has long been awaited, quantitative, comprehensive, and spatiotemporal measurements have posed analytic challenges. The recent development of novel approaches for studying mitochondrial protein synthesis has overcome these issues and expands our understanding of the unique translation system. Here, we review the current technologies for the investigation of mitochondrial translation and the insights provided by their application.

Keywords: FUNCAT; Mitochondria; Mitoribosome; Ribosome profiling; Translation

DOI: https://doi.org/10.1093/jb/mvac005

Curr Protoc. 2022 Jan;2(1): e360

Chromatin Immunoprecipitation in Chloroplasts.

Wenjie Wang, Lingling Cheng, Qianwen Sun.

Chromatin is the genetic material assembled by nucleic acids (including DNA and RNA) and proteins. The biological functions of chromatin are highly dependent on the interaction between DNA (and/or RNA) and proteins that bind to it. Chromatin immunoprecipitation (ChIP) is a powerful technique for evaluating these interactions and has been widely used to characterize the functions of nuclear proteins. However, its application in identifying plant organellar chromatin-binding proteins is lagging. This article describes the method for analyzing the association of chloroplast-localized proteins with the chloroplast genome. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Chloroplast isolation Basic Protocol 2: Crosslinking of DNA-Protein complexes Basic Protocol 3: Chromatin isolation and preparation Support Protocol: Bead-antibody complex preparation Basic Protocol 4: Immunoprecipitation and washes Basic Protocol 5: DNA preparation Basic Protocol 6: Analysis of results.

Keywords: chloroplast; chromatin immunoprecipitation; genome

DOI: https://doi.org/10.1002/cpz1.360

DNA Repair (Amst). 2022 Jan 13. pii: S1568-7864(22)00002-7. [Epub ahead of print]110 103273

Large dataset of octocoral mitochondrial genomes provides new insights into mt-mutS evolution and function.

Viraj Muthye, Cameron D Mackereth, James B Stewart, Dennis V Lavrov.

All studied octocoral mitochondrial genomes (mt-genomes) contain a homologue of the Escherichia coli mutS gene, a member of a gene family encoding proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life, as well as viruses, octocoral mt-mutS is the only such gene found in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of several characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. 2020 study [Nature Ecology & Evolution 4:1531-1538] to assemble complete mt-genomes for 94 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mt-genomes, which we used to re-analyze the conservation and evolution of mt-mutS. In our analysis, we discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus spp. assembled from Quattrini et al. data. This species displayed accelerated rate and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals.

Keywords: DNA repair; Mismatch repair; Mitochondria; MutS; Octocoral

DOI: https://doi.org/10.1016/j.dnarep.2022.103273

Front Mol Biosci. 2021 ;8 815845

When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer.

Denisse Carvajal-Maldonado, Lea Drogalis Beckham, Richard D Wood, Sylvie Doublié.

DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3'-5' exonuclease, 5' nuclease, or end-trimming nuclease) or lyase (5' dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.

Keywords: DNA polymerases; DNA repair; lyase activity; nuclease activity; nucleotidyl transfer; proofreading

DOI: https://doi.org/10.3389/fmolb.2021.815845

Bio Protoc. 2021 Dec 20. 11(24): e4269

Single Molecular Resolution to Monitor DNA Replication Fork Dynamics upon Stress by DNA Fiber Assay.

Wenpeng Liu.

DNA replication always encounters numerous endogenous and exogenous stresses during the cell cycle. Measuring the cell responses to stress has primarily relied on cell survival and incorporation of radioactive dNTPs, which is limited in resolution. A higher resolution is required to monitor how replication and repair respond to these stresses. This protocol describes a procedure to monitor the length of new synthesized DNA in a single molecular resolution called DNA fiber assay. The fiber assay relies on labeling of nascent DNA with the nucleoside analog 5-Chloro-2'-deoxyuridine (CldU) and 5-Iodo-2'-deoxyuridine (IdU). We can visualize the labeled nascent DNA in single molecular resolution by immunostaining. By measuring labeled DNA length, the assay permits interrogation of replication speed at given conditions, end processing at the reversed fork, and fork restart after repair.

Keywords: DNA fiber assay; Fork degradation; Fork restart; Fork speed; Nascent DNA strand degradation

DOI: https://doi.org/10.21769/BioProtoc.4269