Biology subjects

Nagaraju, G.

Publications and source records attributed to Nagaraju, G..

3 recordsLinked to original sources

bioRxiv2026

Distinct complexes of RAD51 paralogs participate in different fork protection pathways

Error-free genome duplication is critical for cellular homeostasis and genome maintenance. Stalled forks undergo remodeling during replication stress, and unprotected forks undergo degradation by nucleases. RAD51 paralogs are evolutionarily conserved essential proteins for genome maintenance with diverse roles ranging from homologous recombination (HR) to replication stress responses. However, the mechanisms underlying their participation in fork maintenance remain unclear. Here, we demonstrate that the RAD51 paralogs do not participate in the canonical SMARCAL1-BRCA2 axis of fork protection. Instead, RAD51D-XRCC2 (DX2) and RAD51C-XRCC3 (CX3) complexes protect forks remodeled by the FBH1 helicase, whereas the RAD51B-RAD51C (BC) subcomplex but not BRCA2 safeguards forks remodeled by the FANCM translocase, revealing a new FANCM-mediated pathway of fork remodeling which is protected by the BC sub-complex. Mechanistically, we show that FANCM-mediated fork reversal is RAD51-dependent and generates substrates for MRE11-, EXO1-, and DNA2-mediated degradation in the absence of RAD51B/C. Our findings establish the participation of the RAD51 paralogs in multiple fork protection pathways in a fork-remodeler-specific manner, highlighting the existence of several independent fork remodeling and protection mechanisms for maintaining genomic stability under replication stress.

cell biology↗

bioRxiv2024

RNF20-mediated H2B monoubiquitination protects stalled forks and promotes fork restart

Chromatin modifications play an important role in transcription, DNA replication and repair. Nonetheless, whether histone modifications regulate replication stress responses remains obscure. Here, we show that RNF20 localizes to and promotes H2B monoubiquitination (H2Bub) at replicating sites. Knockdown of RNF20 leads to degradation of stalled forks by MRE11 nuclease, which can be rescued by inhibition of MRE11 and co-depletion of SMARCAL1/HLTF/ZRANB3 fork remodelers. RNF20 facilitates the loading of RAD51 and RAD51C at the stalled fork sites and participates in the same pathway of RAD51/RAD51C-mediated fork protection and restart. Analyses with the RING domain and phosphorylation-deficient mutants of RNF20 showed that its catalytic activity and ATR/ATM-mediated phosphorylation are essential for its role in replication stress responses. Notably, treatment of RNF20-depleted cells with chromatin relaxing agents rescue the fork protection and restart defects. Collectively, our studies uncover the role of RNF20-mediated H2Bub in regulating the chromatin dynamics to safeguard the replicating genomes.

molecular biology↗

bioRxiv2020

Nitric oxide-inducible Fe-S cluster biogenesis enables Mycobacterium tuberculosis to persist in mice

The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtbs physiology are not fully understood. Since NO damages Fe-S clusters of essential enzymes, the mechanism(s) involved in regulating iron-sulfur (Fe-S) cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA- sequencing of Mtb{Delta}sufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of Mtb {Delta}sufR. Lastly, Mtb{Delta} sufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics. HighlightsO_LIMycobacterium tuberculosis (Mtb) induces the expression of suf operon for Fe-S cluster biogenesis in response to nitric oxide (NO). C_LIO_LIWe found that a transcription factor SufR senses NO via its 4Fe-4S cluster and regulates the expression of the suf operon for Fe-S cluster biogenesis. C_LIO_LISufR-regulated Fe-S cluster biogenesis confers respiratory and redox features that promote recovery of Mtb from NO stress. C_LIO_LISufR activity is required to support the NO-dependent persistence of Mtb in macrophages and mice. C_LI

microbiology↗