Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability.

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.

UvrB protein of Corynebacterium pseudotuberculosis complements the phenotype of knockout Escherichia coli and recognizes DNA damage caused by UV radiation but not 8-oxoguanine in vitro.

In prokaryotic cells, the UvrB protein plays a central role in nucleotide excision repair, which is involved in the recognition of bulky DNA lesions generated by chemical or physical agents. The present investigation aimed to characterize the uvrB gene of Corynebacterium pseudotuberculosis (CpuvrB) and evaluate its involvement in the DNA repair system of this pathogenic organism. In computational analysis, the alignment of the UvrB protein sequences of Escherichia coli, Mycobacterium tuberculosis, Bacillus caldotenax and Corynebacterium pseudotuberculosis showed high similarity and the catalytic amino acid residues and functional domains are preserved. A CpUvrB model was constructed by comparative modeling and presented structural similarity with the UvrB of E. coli. Moreover, in molecular docking analysis CpUvrB showed favorable interaction with EcUvrA and revealed a preserved ATP incorporation site. Heterologous functional complementation assays using E. coli uvrB-deficient cells exposed to UV irradiation showed that the CpUvrB protein contributed to an increased survival rate in relation to those in the absence of CpUvrB. Damaged oligonucleotides containing thymine dimer or 8-oxoguanine lesion were synthesized and incubated with CpUvrB protein, which was able to recognize and excise UV irradiation damage but not 8-oxoguanine. These results suggest that CpUvrB is involved in repairing lesions derived from UV light and encodes a protein orthologous to EcUvrB.