Biology subjects

Carvajal-Garcia, J.

Publications and source records attributed to Carvajal-Garcia, J..

7 recordsLinked to original sources

bioRxiv2026

Polynucleotide Phosphorylase Stops Replication Restart by Degrading the RNA within R-loops during Replication-Transcription Conflicts

DNA replication and transcription machineries function simultaneously, on the same template, leading to conflicts between the two. During conflicts, the nascent mRNAs can hybridize to their template strands, generating RNA:DNA hybrids, which reside within nucleic acid structures known as R-loops. Accumulation of R-loops can lead to severe replication fork stalling, eventually leading to cell death. In bacteria, there are well-known replication restart mechanisms that should rescue these stalled replication forks, and yet, during conflicts, this process is inhibited. Additionally, in vitro work has shown that RNA:DNA hybrids are substrates for replication restart. Here, we discovered that the highly conserved exonuclease Polynucleotide Phosphorylase (PNPase in Bacillus subtilis) prevents replication restart from RNA:DNA hybrids, specifically at regions of severe conflicts where R-loops accumulate. Our in vitro data show that PNPase binds RNA:DNA hybrids and digests the RNA in these structures. Consistently, we found that in vivo, PNPase binds to conflict regions and reduces R-loop levels. To our knowledge, the only other class of enzymes known to digest the RNA from hybrids are RNases H. Our findings identify PNPase as a new enzyme that can perform a similar function. Importantly, our data show that PNPase inhibits replication restart from R-loops and its absence allows for replication restart from conflict regions. We also observed that PNPase activity reduces mutations, suggesting that replication restart from RNA:DNA hybrids is highly mutagenic. We propose that PNPase acts as a safeguard against mutagenic replication restart from RNA:DNA hybrids within R-loops by digesting the RNA which could re-initiate replication.

genetics↗

bioRxiv2026

Processing of Reversed Replication Forks is Required for the Resolution of Replication-Transcription Conflicts

DNA replication and transcription occur simultaneously on the same template, leading to conflicts between the two machineries. Conflicts stall replication forks, lead to genome instability, mutagenesis, and the formation of deleterious R-loop structures. There are two types of conflicts: depending on the strand where a gene is encoded, the two machineries either meet head-on or co-directionally. The adverse outcomes of conflicts in the head-on orientation are significantly more detrimental to cells compared to co-directional conflicts. Despite many studies across various organisms, how the replication fork structure is impacted by these encounters remains unclear. Here, we performed an unbiased genetic screen using a transposon library to identify factors essential for surviving head-on conflicts in Bacillus subtilis. Our screen identified three hits: RNase HIII, AddA and AddB. Our prior work had shown that RNase HIII, which processes R-loops, is essential for surviving head-on conflicts. However, AddA and AddB, which function together as a complex to process blunt DNA ends, have not been previously identified as essential conflict resolution factors. Through follow-up genetic and biochemical analyses, we found that the helicase activity but not the nuclease activity of this complex is required for conflict resolution. Based on the fundamental properties of DNA at replication fork structures, our work collectively indicates that upon head-on conflicts, the nascent strands form a reversed fork structure, which is then unwound by AddAB, which leads to re-annealing to the parental strands. This process re-establishes an intact replication fork that can be used to restart replication after conflicts with transcription.

molecular biology↗

bioRxiv2026

A universal regulatory mechanism for prevention of replication restart from RNA:DNA hybrids

RNA:DNA hybrids form across all genomes. In vitro studies showed that these structures can be used to restart stalled replication forks, especially upon replication-transcription conflicts, however, this has not been tested in vivo. Here, we identify a mechanism that prevents replication restart from hybrids within cells. We identified the tRNA ligase, AsnRS, as a regulator of replication restart from hybrids at conflict regions in Bacillus subtilis. We determined that the DNA translocase Mfd and DNA polymerase PolA play key roles in this pathway. We unraveled a mechanism whereby Mfd removes RNA Polymerases, exposing 3 ends of the RNAs within hybrids. These 3 ends are then capped by AsnRS, preventing restart by PolA. Remarkably, the mammalian homologs of AsnRS, NARS1 and NARS2, fully complement the AsnRS phenotypes in bacteria. We propose that this is a universally conserved mechanism that prevents untimely replication initiation outside of origins from bacteria to humans.

genetics↗

bioRxiv2022

A small molecule that inhibits the evolution of antibiotic resistance

Antimicrobial resistance (AMR) rapidly develops against almost all available therapeutics. New antibiotics target essential processes in bacteria but fail to address the root of the problem: mutagenesis and evolution. We recently proposed that inhibiting the molecular mechanisms underlying bacterial evolution is the ultimate solution to preventing AMR development. Here, we describe the first compound that inhibits the occurrence and progression of AMR by directly targeting a highly conserved bacterial evolvability factor, Mfd. We previously found that this RNA polymerase-associated translocase is required for rapid AMR development across highly divergent pathogens. Through an in vivo screen, we identified 43 potential Mfd-inhibiting compounds. Here we present on target validation, biochemical characterization, and in vivo efficacy studies of a lead compound, referred to as ARM-1. ARM-1 binds Mfd and modulates its RNA polymerase interaction. Inhibition of Mfd activity by ARM-1 delays the development of mutations and resistance acquisition, both in pure culture and during infection. Importantly, our data show that this compound prevents the evolution of AMR across highly divergent pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium. The novel compound we present here has the potential to develop into a clinically useful "anti-evolution" drug. This work demonstrates that the molecular mechanisms of evolution are pharmaceutically targetable, and that this strategy could help prevent AMR development.

microbiology↗

bioRxiv2022

Nucleotide excision repair is universally mutagenic and transcription-associated

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one the main public health problems of the 21st century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics tested and across highly divergent species, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This strongly suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies that a highly conserved pathway drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

molecular biology↗

bioRxiv2020

DNA polymerase theta suppresses mitotic crossing over

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol {theta}) to use resected, 3 single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol {theta} to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol {theta} in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol {theta} and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol {theta} or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol {theta}. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol {theta} can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells. Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

genetics↗

bioRxiv2019

Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta

DNA Polymerase Theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3 single stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3 terminus and favoring bi-directional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3-30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification, and shows both how it mitigates limitations implicit in the microhomology requirement, and generates distinctive genomic scars associated with pathogenic genome instability.

molecular biology↗