Biology subjects

Andres, S. N.

Publications and source records attributed to Andres, S. N..

4 recordsLinked to original sources

bioRxiv2026

Translesion synthesis protein ImuA from Mycolicibacterium smegmatis is a hexameric helicase-nuclease

Translesion DNA synthesis (TLS) enables DNA replication across damaged DNA and promotes stress-induced mutagenesis that contributes to antibiotic resistance in bacteria. The conserved ImuABC mutasome is essential for TLS in many bacterial species, yet the molecular function of its accessory protein, ImuA, has remained elusive. Here we show that Mycolicibacterium smegmatis ImuA assembles into a hexameric complex, likely arranged as a dimer of trimers, with dual enzymatic activities that reshape current models of its role in DNA damage tolerance. We show that ImuA functions as an ATP-dependent helicase that preferentially unwinds DNA substrates containing single-stranded DNA overhangs and identify amino acids required for both hexamer formation and helicase activity. Unexpectedly, ImuA also possesses ATP-independent 5' exonuclease activity, selectively processing ssDNA substrates with free 5' ends. We show a basic patch on the N-terminus is essential for stabilizing both the nuclease motif and oligomerization. Together, these findings identify ImuA as an active DNA-processing enzyme rather than a passive accessory factor and establish oligomerization as a prerequisite for its function. Our work provides a mechanistic framework for understanding how ImuA may function within the ImuABC mutasome to coordinate DNA processing during translesion synthesis.

biochemistry↗

bioRxiv2025

The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions

Bacterial non-homologous end joining (NHEJ) is a DNA double-strand break (DSB) repair pathway that relies on the Ku-LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminus has been highlighted for its role in LigD recruitment to DNA DSBs and stimulation of ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences other LigD activities. Here, we combine NMR spectroscopy, structural modelling and mutational analysis to define the interaction interface between the Ku C-terminus and LigD. We identify critical residues in Ku (E246, V248, S258, K260, and N266) and the LigD polymerase (D162, V194, R198) and ligase (D522, K579, L580) domains that mediate this interaction. Functional assays reveal that Ku stimulates LigD ligase activity through contacts with both polymerase and ligase domains, while Ku attenuates template-dependent polymerase activity, contrasting previous studies with Pseudomonas aeruginosa homologs. Disrupting the Ku-LigD interface, either through Ku or LigD mutations, abolishes ligase stimulation and restores polymerase activity, highlighting a dual regulatory mechanism. Our data supports a model where Kus C-terminal region forms a bipartite interface with LigD to balance repair activity. These findings provide mechanistic insight into the Ku-LigD repair mechanism and uncovers species-specific differences in bacterial NHEJ. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/693941v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@802e3org.highwire.dtl.DTLVardef@1f5ac14org.highwire.dtl.DTLVardef@1f46677org.highwire.dtl.DTLVardef@7312b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Poly ADP-Ribose Signaling is Dysregulated in Huntington Disease Patients

Huntington disease (HD) is a genetic neurodegenerative disease caused by CAG expansion in the Huntingtin (HTT) gene, translating to an expanded polyglutamine tract in the huntingtin (HTT) protein. Age at disease onset correlates to CAG repeat length but varies by decades between individuals with identical repeat lengths. Genome-wide association studies link HD modification to DNA repair and mitochondrial health pathways. Clinical studies show elevated DNA damage in HD, even at the premanifest stage. A major DNA repair node influencing neurodegenerative disease is the PARP pathway. Accumulation of poly ADP-ribose (PAR) has been implicated in Alzheimer and Parkinson diseases, as well as cerebellar ataxia. We report that HD mutation carriers have lower cerebrospinal fluid PAR levels than healthy controls, starting at the premanifest stage. Human HD iPSC-derived neurons and patient- derived fibroblasts have diminished PAR response in the context of elevated DNA damage. We have defined a PAR-binding motif in huntingtin, detected huntingtin complexed with PARylated proteins in human cells during stress, and localized huntingtin to mitotic chromosomes upon inhibition of PAR degradation. Direct huntingtin PAR binding was measured by fluorescence polarization and visualized by atomic force microscopy at the single molecule level. While wild type and mutant huntingtin did not differ in their PAR binding ability, purified wild type huntingtin protein increased in vitro PARP1 activity while mutant huntingtin did not. These results provide insight into an early molecular mechanism of HD, suggesting possible targets for the design of early preventive therapies. Significance statementA consensus on dysfunctional DNA repair has emerged in neurodegenerative disease research, with elevated poly ADP-ribose (PAR) signaling more recently implicated. In contrast, we have identified a deficient PAR response in Huntingtons disease (HD) patient spinal fluid samples and cells. This may be explained by the inability of huntingtin protein bearing the HD-causing mutation to stimulate production of PAR the way the wild type protein does. Since drugs that target PAR production and degradation have already been developed, these findings present an exciting avenue for therapeutic intervention for HD.

neuroscience↗

bioRxiv2021

Interplay between nucleoid-associated proteins and transcription factors in controlling specialized metabolism in Streptomyces

Lsr2 is a small nucleoid-associated protein found throughout the actinobacteria. Lsr2 functions similarly to the well-studied H-NS, in that it preferentially binds AT-rich sequences and represses gene expression. In Streptomyces venezuelae, Lsr2 represses the expression of many specialized metabolic clusters, including the chloramphenicol antibiotic biosynthetic gene cluster, and deleting lsr2 leads to significant upregulation of chloramphenicol cluster expression. We show here that Lsr2 likely exerts its repressive effects on the chloramphenicol cluster by polymerizing along the chromosome, and by bridging sites within and adjacent to the chloramphenicol cluster. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not upregulated in an lsr2 mutant strain. We demonstrate that CmlR is essential for chloramphenicol production, and further reveal that CmlR functions to counter-silence Lsr2s repressive effects by recruiting RNA polymerase and enhancing transcription, with RNA polymerase effectively clearing bound Lsr2 from the chloramphenicol cluster DNA. Our results provide insight into the interplay between opposing regulatory proteins that govern antibiotic production in S. venezuelae, which could be exploited to maximize the production of bioactive natural products in other systems. IMPORTANCESpecialized metabolic clusters in Streptomyces are the source of many clinically-prescribed antibiotics. However, many clusters are not expressed in the laboratory due to repression by the nucleoid-associated protein Lsr2. Understanding how Lsr2 represses cluster expression, and how repression can be alleviated, are key to accessing the metabolic potential of these bacteria. Using the chloramphenicol biosynthetic cluster from Streptomyces venezuelae as a model, we explored the mechanistic basis underlying Lsr2-mediated repression, and activation by the pathway-specific regulator CmlR. Lsr2 polymerized along the chromosome and bridged binding sites located within and outside of the cluster, promoting repression. Conversely, CmlR was essential for chloramphenicol production, and further functioned to counter-silence Lsr2 repression by recruiting RNA polymerase and promoting transcription, ultimately removing Lsr2 polymers from the chromosome. Manipulating the activity of both regulators led to >130x increase in chloramphenicol levels, suggesting that combinatorial regulatory strategies can be powerful tools for maximizing natural product yields.

microbiology↗