No, this is not a new movie in the Marvel Cinematic Universe, but the implications might be enough for Thanos to even raise an eyebrow. The newly published study takes a hard look at the underlying mechanisms for the DNA mismatch repair (MMR) pathway—which helps maintain genomic fidelity by more than 1000-fold. Typically, MMR works in concert with the DNA replication machinery, utilizing two proofreading proteins that work together as an emergency stop button to prevent replication errors. Now, new research from North Carolina (NC) State University and the University of North Carolina (UNC) at Chapel Hill shows how these proteins—MutL and MutS—prevent DNA replication errors by creating an immobile structure that recruits more proteins to the site to repair the error.

“Although mismatches are rare, the human genome contains approximately six billion nucleotides in every cell, resulting in approximately 600 errors per cell, and the human body consists of more than 37 trillion cells,” explained co-senior study investigator Dorothy Erie, PhD, a chemistry professor at UNC-Chapel Hill and a member of UNC’s Lineberger Comprehensive Cancer Center. “Consequently, if these errors go unchecked, they can result in a vast array of mutations, which in turn can result in a variety of cancers, collectively known as Lynch Syndrome.”

Findings from the new study—published recently in two PNAS articles titled, “Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby” and “Dynamic human MutSα-MutLα complexes compact mismatched DNA”—also suggest that the immobile structure could also prevent the mismatched region from being “packed” back into the cell during division.

When a cell prepares to divide, the DNA splits, with the double helix “unzipping” into two separate backbones. New nucleotides are filled into the gaps on the other side of the backbone, pairing with their counterparts and replicating the DNA to make a copy for both the old and the new cells. The nucleotides are a correct match most of the time, but occasionally—about one time in 10 million—there is a mismatch.

A pair of proteins known as MutS and MutL work together to initiate repair of these mismatches. MutS slides along the newly created side of the DNA strand after it is replicated, proofreading it. When it finds a mismatch, it locks into place at the site of the error and recruits MutL to come and join it. MutL marks the newly formed DNA strand as defective and signals a different protein to gobble up the portion of the DNA containing the error. Then the nucleotide matching starts over, filling the gap again. The entire process reduces replication errors around a 1000-fold, serving as one of our body’s best defenses against genetic mutations that can lead to cancer.

“We know that MutS and MutL find, bind, and recruit repair proteins to DNA,” noted co-senior study investigator, Keith Weninger, PhD, a biophysicist and university faculty scholar at NC State. “But one question remained—do MutS and MutL move from the mismatch during the repair recruiting process, or stay where they are?”

Between the two papers, Weninger and Erie looked at both human and bacterial DNA to gain a clearer temporal and structural picture of what happens when MutS and MutL engage in mismatch repair.

Using both fluorescent and non-fluorescent imaging techniques, including atomic force microscopy, optical spectroscopy, and tethered particle motion, the researchers found that MutL “freezes” MutS in place at the site of the mismatch, forming a stable complex that stays in that vicinity until the repair can take place. The complex appears to reel in the DNA around the mismatch as well, marking and protecting the DNA region until the repair can occur.

“We use single-molecule FRET to follow the post-recognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA,” the authors wrote. “Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop the movement of the mobile clamp on DNA. MutS’s frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL’s ability to trap MutS, should increase the probability that MutS–MutL MMR initiation complexes localize near the mismatch.”

“Due to the mobility of these proteins, current thinking envisioned MutS and MutL sliding freely along the mismatched strand, rather than stopping,” Weninger concluded. “This work demonstrates that the process is different than previously thought. Additionally, the complex’s interaction with the strand effectively stops any other processes until repair takes place. So, the defective DNA strand cannot be repacked into a chromosome and then carried forward through cell division.”

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