Biology subjects

Lim, H. C.

Publications and source records attributed to Lim, H. C..

5 recordsLinked to original sources

bioRxiv2026

RecBCD Prevents Bacterial Autoimmunity by Clearing Self-DNA Ends Produced During Replication Termination

Viral-induced DNA damage triggers both eukaryotic and prokaryotic innate immunity. Here, we report that self-DNA ends generated routinely during normal termination of bacterial replication suffice to trigger the P2-encoded OLD antiphage system. Normally, these physiological triggers are cleared by the dual helicase-nuclease activities of the conserved RecBCD enzyme, which acts as an immune safeguard analogous to mammalian TREX1 exonuclease and plant DBR1 debranching enzyme. However, when this clearance fails, the transient self-DNA ends persist and are recognized by ATP-bound OLD dimers via a positively charged channel across their ATPase subunits. ATP hydrolysis then activates OLD to cleave tRNAs, causing cytotoxicity. Single-cell microscopy reveals that dozens of OLD molecules associate with kilobases of adjacent DNA, amplifying the sparse physiological DNA end signal. Our findings reveal how an endogenous byproduct poses a constant autoimmune threat in bacteria, highlighting a universal molecular conflict across kingdoms and the convergent solutions evolved to resolve it.

microbiology↗

bioRxiv2025

Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli

Certain phages and microbes use Diversity-Generating Retroelements (DGRs) for targeted adaptation via a process called mutagenic retrohoming. This process relies on an error-prone reverse transcriptase that specifically misincorporates bases opposite adenines in the RNA template. The resulting mutagenized complementary DNA then integrates into a homologous target gene to introduce mutations at adenine residues. Although the functions of core DGR components are well-defined, we still dont know how other cellular factors influence their activity. To address this, we reconstituted the DGR from the Bordetella phage BPP-1 in Escherichia coli. Our systematic analysis uncovered several cellular factors that influence its activity. By sequencing the cDNA, we discovered the reverse transcriptase is particularly mutagenic in the 5-AAC-3 and 5-ACC-3 contexts, which explains their abundance in natural DGR templates. A transposon sequencing screen identified both positive and negative regulators, including the single-stranded DNA exonuclease ExoI, which unexpectedly inhibits DGR activity through a nuclease-independent mechanism. Using a massively parallel reporter assay, we found that DGR preferentially edits targets located near the replication origin and co-directionally oriented with replication. In the preferred orientation, the target sequence is located on the lagging-strand template, where the extended single-stranded DNA gap likely facilitates base pairing with the incoming complementary DNA. Collectively, these findings advance our understanding of how DGRs function within the cellular context and enabled a 1000-fold improvement in editing efficiency, establishing a foundation for future targeted-mutagenesis applications.

microbiology↗

bioRxiv2023

HBV rewires liver cancer signaling by altering PP2A complexes

Infection by hepatitis B virus (HBV) increases risk for liver cancer by inducing inflammation, cellular stress and cell death. To elucidate the molecular pathways by which HBV promotes cancer development and progression, we used affinity purification mass spectrometry to comprehensively map a network of 145 physical interactions between HBV and human host proteins in hepatocellular carcinoma (HCC). We find that viral proteins target host factors that are preferentially mutated in non-HBV-associated HCC, implicating cancer pathways whose interaction with HBV plays a role in HCC. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we show that HBx remodels the PP2A phosphatase complex, altering its effect on tumor signaling. HBx excludes striatin-family regulatory subunits from PP2A, causing Hippo kinase activation and unmasking a requirement for mTOR complex 2 to maintain expression of the YAP oncoprotein in HCC. Thus, HBV rewires HCC to expose potentially targetable signaling dependencies. SignificancePrecision medicine has revolutionized cancer treatment but remains elusive for HCC. We used proteomics to define HBV/host interactions and integrated them with HCC mutations. The results implicate modifiers of HCC behavior via remodeling of host complexes and illuminate new biological mechanisms in advanced disease for therapeutic investigation.

cancer biology↗

bioRxiv2018

Sidewall cell envelope synthesis and remodeling in pole-growing mycobacteria

O_SCPLOWDC_SCPLOW-amino acid probes label cell wall peptidoglycan at both the poles and sidewall of pole-growing mycobacteria. Since peptidoglycan assembly along the cell periphery could provide a rapid, growth-independent means by which to edit the cell wall, we sought to clarify the precise metabolic fates of these probes. O_SCPLOWDC_SCPLOW-amino acid monopeptides were incorporated into peptidoglycan by O_SCPLOWLC_SCPLOWO_SCPCAP,C_SCPCAPO_SCPLOWDC_SCPLOW-transpeptidase remodeling enzymes to varying extents. Dipeptides were incorporated into cytoplasmic precursors. While dipeptide-marked peptidoglycan synthesis at the poles was associated with cell elongation, synthesis along the periphery was highly responsive to cell wall damage. Our observations suggest a post-expansion role for peptidoglycan assembly along the mycobacterial sidewall and provide a conceptual framework for understanding cell wall robustness in the face of polar growth.

microbiology↗

bioRxiv2018

Maturing Mycobacterial Peptidoglycan Required Non-canonical Crosslinks to Maintain Shape

In most well studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin binding proteins (PBPs). However, in mycobacteria, L,D-transpeptidase (LDT)-mediated crosslinks are highly abundant. To elucidate the role of these unusual crosslinks, we characterized mycobacterial cells lacking all LDTs. We find that LDT-mediated crosslinks are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT-inhibition. Thus, knowledge about the single-cell distribution of drug targets can be exploited to more effectively treat this pathogen.

microbiology↗