A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples.

Mutational signatures of complex genomic rearrangements in human cancer

Complex genomic rearrangements (CGRs) are common in cancer and are known to form via two aberrant cellular structures—micronuclei and chromatin bridge. However, which mechanism is more relevant to CGR formation in cancer cells and whether there are other undiscovered mechanisms remain open questions. Here, we analyze 2,014 CGRs from 2,428 whole-genome sequenced tumors and deconvolute six CGR signatures based on the topology of CGRs. Through rigorous benchmarking, we show that our CGR signatures are highly accurate and biologically meaningful. Three signatures can be attributed to known biological processes—micronuclei- and chromatin-bridge-induced chromothripsis and extrachromosomal DNA. More than half of the CGRs belong to the remaining three newly discovered signatures. A unique signature (we named “hourglass chromothripsis”) with highly localized breakpoints and small amount of DNA loss is abundant in prostate cancer. Through genetic association analysis, we find SPOP as a candidate gene causing hourglass chromothripsis and playing important role in maintaining genome integrity. Our study offers valuable insights into the formation of CGRs.

Mutational signatures of complex genomic rearrangements in human cancer

Complex genomic rearrangements (CGRs) are common in cancer and are known to form via two aberrant cellular structures-micronuclei and chromatin bridge. However, which mechanism is more relevant to CGR formation in cancer cells and whether there are other undiscovered mechanisms remain open questions. Here, we analyze 2,014 CGRs from 2,428 whole-genome sequenced tumors and deconvolute six CGR signatures based on the topology of CGRs. Through rigorous benchmarking, we show that our CGR signatures are highly accurate and biologically meaningful. Three signatures can be attributed to known biological processes-micronuclei- and chromatin-bridge-induced chromothripsis and extrachromosomal DNA. More than half of the CGRs belong to the remaining three newly discovered signatures. A unique signature (we named "hourglass chromothripsis") with highly localized breakpoints and small amount of DNA loss is abundant in prostate cancer. Through genetic association analysis, we find SPOP as a candidate gene causing hourglass chromothripsis and playing important role in maintaining genome integrity. Our study offers valuable insights into the formation of CGRs.

Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex

During genome duplication, the replication fork encounters a plethora of obstacles in the form of damaged bases, DNA–cross-linked proteins, and secondary structures. How cells protect DNA integrity at sites of stalled replication is currently unknown. Here, by engineering “primase deserts” into the Caenorhabditis elegans genome close to replication-impeding G-quadruplexes, we show that de novo DNA synthesis downstream of the blocked fork suppresses DNA loss. We next identify the pol α-primase complex to limit deletion mutagenesis, a conclusion substantiated by whole-genome analysis of animals carrying mutated POLA2/DIV-1. We subsequently identify a new role for the 9-1-1 checkpoint clamp in protecting Okazaki fragments from resection by EXO1. Together, our results provide a mechanistic model for controlling the fate of replication intermediates at sites of stalled replication.

Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis

Thymineless death in Escherichia coli thyA mutants growing in the absence of thymidine (dT) is preceded by a substantial resistance phase, during which the culture titer remains static, as if the chromosome has to accumulate damage before ultimately failing. Significant chromosomal replication and fragmentation during the resistance phase could provide appropriate sources of this damage. Alternatively, the initial chromosomal replication in thymine (T)-starved cells could reflect a considerable endogenous dT source, making the resistance phase a delay of acute starvation, rather than an integral part of thymineless death. Here we identify such a low-molecular-weight (LMW)-dT source as mostly dTDP-glucose and its derivatives, used to synthesize enterobacterial common antigen (ECA). The thyA mutant, in which dTDP-glucose production is blocked by the rfbA rffH mutations, lacks a LMW-dT pool, the initial DNA synthesis during T-starvation and the resistance phase. Remarkably, the thyA mutant that makes dTDP-glucose and initiates ECA synthesis normally yet cannot complete it due to the rffC defect, maintains a regular LMW-dT pool, but cannot recover dTTP from it, and thus suffers T-hyperstarvation, dying precipitously, completely losing chromosomal DNA and eventually lysing, even without chromosomal replication. At the same time, its ECA+thyA parent does not lyse during T-starvation, while both the dramatic killing and chromosomal DNA loss in the ECA-deficient thyA mutants precede cell lysis. We conclude that: 1) the significant pool of dTDP-hexoses delays acute T-starvation; 2) T-starvation destabilizes even nonreplicating chromosomes, while T-hyperstarvation destroys them; and 3) beyond the chromosome, T-hyperstarvation also destabilizes the cell envelope.

Programmed DNA Elimination in Vertebrates

Over the last few decades, an increasing number of vertebrate taxa have been identified that undergo programmed genome rearrangement, or programmed DNA loss, during development. In these organisms, the genome of germ cells is often reproducibly different from the genome of all other cells within the body. Although we clearly have not identified all vertebrate taxa that undergo programmed genome loss, the list of species known to undergo loss now represents ∼10% of vertebrate species, including several basally diverging lineages. Recent studies have shed new light on the targets and mechanisms of DNA loss and their association with canonical modes of DNA silencing. Ultimately, expansion of these studies into a larger collection of taxa will aid in reconstructing patterns of shared/independent ancestry of programmed DNA loss in the vertebrate lineage, as well as more recent evolutionary events that have shaped the structure and content of eliminated DNA. Expected final online publication date for the Annual Review of Animal Biosciences, Volume 9 is February 16, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast

SUMMARYNaturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not timely repaired. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including topoisomerase-1 trapped in covalent crosslinks (Top1ccs). Here we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S-phase dependent manner to assist eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitize cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among potential Ddi1 targets we found the core component of RNAP II and show that its genotoxin-induced degradation is impaired in ddi1. Together, we propose that the Ddi1 protease contributes to DPC proteolysis.