DNA mismatch repair protein, MutS Homolog 3 (MSH3) is a human homologue of the bacterial mismatch repair protein MutS that participates in the mismatch repair (MMR) system. MSH3 typically forms the heterodimer MutSβ with MSH2 in order to correct long insertion/deletion loops and base-base mispairs in microsatellites during DNA synthesis. Deficient capacity for MMR is found in approximately 15% of colorectal cancers, and somatic mutations in the MSH3 gene can be found in nearly 50% of MMR-deficient colorectal cancers.

In humans, the encoding gene for MSH3 is found on chromosome 5 at location 5q11-q12 upstream of the dihydrofolate reductase (DHFR) gene. MSH3 is encoded by 222,341 base pairs and creates a protein consisting of 1137 amino acids.

MSH3 is typically expressed at low levels in several transformed cell lines—including HeLa, K562, HL-60, and CEM—as well as a large range of normal tissues including spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, lung, liver, skeletal muscle kidney, and pancreas. Although expression levels of MSH3 vary slightly from tissue to tissue, its widespread low-level expression indicates that it is a "housekeeping" gene commonly expressed in all cells.

Over-expression of MSH3 decreased capacity for MMR. When MSH3 is over expressed, drastic changes occur in the relative levels of formation of MutSβ at the expense of MutSα. MutSα is responsible for base-base mispairs and short insertion/deletion loops, while MutSβ repairs long insertion/deletion loops in DNA. A drastic shift in the relative levels of these protein complexes can lead to diminished capacity for MMR. In the case of MSH3 overexpression, MSH2 preferentially heterodimerizes with MSH3 leading to high levels of MutSβ and degradation of the partnerless MSH6 protein which normally complexes with MSH2 to form MutSα.

MSH3 has been shown to interact with MSH2, PCNA, and BRCA1. These interactions form protein complexes that are typically involved in tumor suppression and DNA repair activities.

The primary interaction of MSH3 involves forming the MutSβ complex with MSH2. MutSβ forms as a heterodimer of MSH2 and MSH3 with two primary interaction regions: an amino-terminal region and a carboxy-terminal region. The N-terminal region of MSH3 (amino acids 126-250) contact the N-terminal region of MSH2 aa 378-625. The C-terminal regions connect at aa 1050-1128 of MSH3 and aa 875-934 of MSH2. The binding regions on MSH2 are identical when binding to either MSH3 or MSH6. Adenine nucleotide binding regions in MSH3 and MSH2 are not contained in either of the interaction regions involved in dimerization, allowing MutSβ to bind to DNA and perform MMR.

Proliferating cell nuclear antigen (PCNA) is a protein involved in post-replication MMR. It has been shown that PCNA binds to the MutSβ heterodimer via a binding motif in the N-terminal domain of MSH3. Bound PCNA then localizes the MutSβ complex to replication foci, indicating that PCNA assists in initiating repair by guiding MutSβ and other repair proteins to free termini in recently replicated DNA.

The primary function of MSH3 is to maintain the stability of the genome and enact tumor suppression by forming the heterodimer MutSβ to correct long insertion/deletion loops and base-base mispairs. In the case of long insertion/deletion loops, DNA is severely bent and downstream basepairs can become unpaired and exposed. MutSβ recognizes insertion/deletion loops of 1-15 nucleotides; binding to insertion/deletion loops is achieved by inserting the mismatch-binding domain of MSH3 and part of the mismatch-binding domain of MSH2 into the groove formed by the extreme bend in DNA formed by the insertion/deletion loop.