Abstract Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.

A team of researchers from the Department of Biological Sciences at UNIST has achieved a significant breakthrough in understanding how brain proteins can help alleviate complications arising from DNA replication stress. This groundbreaking discovery holds immense potential for advancing treatments for various diseases, including cancer, neurological disorders, and age-related conditions that result from disruptions in DNA replication.

Led by Professor Jayil Lee, Professor Jang Hyun Choi, and Professor Hongtae Kim, this collaborative effort has unveiled crucial insights into the intricate processes involving NSMF proteins when confronted with DNA replication stress—a fundamental aspect of cellular functioning.

N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, plays a novel role in neuronal development, regulation of movement, reproductive hormone secretion, and olfactory perception. Dysfunctions in this protein can lead to rare conditions like Kallmann syndrome—an affliction characterized by impaired reproductive function and loss of sense of smell.

Figure 1: A schematic diagram illustrating the increase in RPA phosphorylation of ATR by NSMF during DNA replication stress.

In their study focused on alleviating DNA replication stress using NSMF proteins, the research team observed that when protein synthesis encounters obstacles during replication due to various factors causing stress on DNA structure—resulting in single-stranded regions—the replication protein A (RPA) binds uniquely to these exposed single strands. The combined RPA molecules then undergo phosphorylation—a chemical process involving attachment of phosphate groups composed of phosphorus and oxygen. Phosphorylated RPA recruits other proteins that help alleviate replication stress at specific sites along the DNA strand—restoring normal activity.

Interestingly, RPA exhibits weak or strong binding affinity towards single-stranded regions during its interaction with DNA. Through their investigation involving NSMF proteins, the research team discovered that NSMF selectively displaces some weakly bound RPAs while promoting a transition into more stable binding modes for the remaining RPAs. This shift favors an enhanced phosphorylation process, catalyzed by the ataxia telangiectasia and RAD3-related (ATR) kinase—an enzyme crucial for DNA damage response. The team's findings demonstrate that this mechanism accelerates the relief of replication stress.

"This study has significant potential to impact treatments related to cancer, neurological disorders, and age-related conditions by unraveling molecular mechanisms associated with DNA replication," commented Professor Lee.

"Considering NSMF's close relationship with Kallmann syndrome, we anticipate its contribution to advancements in treating this disease," added Yujin Kang, the first author of the study.

Journal Reference
Yujin Kang, Ye Gi Han, Keon Woo Khim, et al., "Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase," Nucleic Acids Res. (2023).