Rabbit Anti-Human MCM2 monoclonal antibody, clone TQ96

Identification of factors associated with human papillomavirus (HPV) cervical histopathology or recurrence/relapse following loop electrosurgical excision procedure (LEEP) would allow for better management of the disease. We investigated whether gene signatures could (i) associate with HPV cervical histopathology and (ii) identify women with post-LEEP disease recurrence/relapse. Gene array analysis was performed on paraffin-embedded cervical tissue-isolated RNA from two cross-sectional cohorts of antiretroviral therapy (ART)-suppressed HIV+HPV+ coinfected women: (i) 55 women in South Africa recruited into three groups: high risk (HR) (-) (n = 16) and HR (+) (n = 15) HPV without cervical histopathology and HR ((+)) HPV with cervical intraepithelial neoplasia (CIN) grade 1/2/3 (n = 24), (ii) 28 women in Botswana with CIN2/3 treated with LEEP 12-month prior to recruitment and presenting with (n = 13) and without (n = 15) lesion recurrence/relapse (tissue was analyzed at first LEEP). Three distinct gene expression signatures identified were able to segregate: (i) HR+HPV and CIN1/2/3, (ii) HR HPV-free and cervical histopathology-free and (iii) HR+ HPV and cervical histopathology-free. Immune activation and neoplasia-associated genes (n = 272 genes; e.g. IL-1A, IL-8, TCAM1, POU4F1, MCM2, SMC1B, CXCL6, MMP12) were a feature of cancer precursor dysplasia within HR HPV infection. No difference in LEEP tissue gene expression was detected between women with or without recurrence/relapse. In conclusion, distinctive gene signatures were associated with presence of cervical histopathology in tissues from ART-suppressed HIV+/HPV+ coinfected women. Lack of detection of LEEP tissue gene signature able to segregate subsequent post-LEEP disease recurrence/relapse indicates additional factors independent of local gene expression as determinants of recurrence/relapse.

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol epsilon and Pol delta, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol e and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol delta and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol epsilon on the lagging strand, and CMG protects Pol epsilon against RFC inhibition on the leading strand. Previous studies show that Pol delta is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol delta-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol delta activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol epsilon binds CMG with a K-d value of 12 nM, but Pol delta binding CMG is undetectable. Pol delta, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol delta-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol delta-CMG interaction, the collision release process is triggered, ejecting Pol delta on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.