hMSH5 Regulates NHEJ and Averts Excessive Nucleotide Alterations at Repair Joints

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions. Inappropriate repair of DSBs can lead to genomic instability, cell death, or cancer.
• Cells employ various DNA repair pathways, such as non-homologous end joining (NHEJ), to maintain genomic integrity.

Purpose of the Study:

• To investigate the role of human MSH5 (hMSH5) in regulating DNA double-strand break repair pathways.
• To elucidate how hMSH5 influences the fidelity of DSB repair, specifically concerning deletions and insertions at repair junctions.

Main Methods:

• RNA interference (RNAi)-mediated knockdown of hMSH5.
• Overexpression of hMSH5.
• Analysis of nucleotide deletions and insertions at DSB repair joints.
• Assessment of microhomology (MH) length at repair junctions.
• Measurement of RPA foci formation as an indicator of single-stranded DNA (ssDNA) presence.
• Evaluation of 53BP1 chromatin spreading to assess DSB end resection.

Main Results:

• hMSH5 down-regulates non-homologous end joining (NHEJ) and restricts DSB end processing.
• Knockdown of hMSH5 resulted in larger nucleotide deletions and insertions, with reduced microhomology (MH) at repair joints.
• hMSH5 overexpression decreased end-joining activity and increased RPA foci, indicating more stable ssDNA.
• Silencing hMSH5 delayed 53BP1 chromatin spreading, correlating with increased DSB end resection.

Conclusions:

• hMSH5 plays a crucial role in maintaining genomic stability by limiting excessive DNA alterations during DSB repair.
• hMSH5 acts as a negative regulator of NHEJ and modulates the extent of DSB end processing, thereby ensuring accurate repair.
• Dysregulation of hMSH5 can contribute to genomic instability by promoting aberrant repair outcomes.