Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53 Å resolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α-helices, five β-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.

Bovine milk and meat factors (BMMFs) are plasmid‐like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co‐markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor‐adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n = 246)], low‐/high‐grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co‐immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor‐adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+/CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD‐adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC‐specific death were increased for higher Rep expression (TMA), with high tumor‐adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF‐specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.