SUMMARY We propose that cell cycle-dependent timing of FEN1 nuclease activity is essential for cell cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S-phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of post-translational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which, in turn, stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination- defective, non-degradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell cycle progression and to prevent transformation.

Design of dual antagonists for the chemokine receptors CCR2 and CCR5 will be greatly facilitated by knowledge of the structural differences of their binding sites. Thus, we computationally predicted the binding site of the dual CCR2/CCR5 antagonist N

We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

The variability within calculated protein residue pKa values calculated using Poisson-Boltzmann continuum theory with respect to small conformational fluctuations is investigated. As a general rule, sites buried in the protein core have the largest pKa fluctuations but the least amount of conformational variability; conversely, sites on the protein surface generally have large conformational fluctuations but very small pKa fluctuations. These results occur because of the heterogeneous or uniform nature of the electrostatic microenvironments at the protein core or surface, respectively. Atypical surface sites with large pKa fluctuations occur at the interfaces between significant anionic and cationic potentials.

Human FEN1 is a structure‐specific DNA nuclease involved in removing 5′ RNA primers of Okazaki fragments and in long‐patch base excision repair. Defects in FEN1 lead to increased genomic instability. Identification of DNA‐binding residues will further elucidate the molecular mechanisms behind genome instability initiation and facilitate cancer therapy linked to hFEN1 mutations. No crystal structure data is available of hFEN1 actively bound to DNA; however, a structure of an hFEN1 homolog, T4 phage RNase H in its active form (pdb 2ihn), is available as well as hFEN1 complexed with PCNA in the inactive form (pdb 1ul1). The 2ihn pdb structure shows 31 DNA‐interacting residues. Computational structural alignment of the two X‐ray crystal structures has revealed 5 residues within the 31 subset of RNase H that have a seemingly structurally conserved equivalent in hFEN1; these are K74, L29, V30, F53, and H174. These conserved equivalents in hFEN1 may therefore interact with DNA. To validate our predictions, we use HPLC to measure specific activity levels of hFEN1 mutant proteins versus the wildtype using fluorescein‐labeled DNA substrate. We found that the specific activities of the mutants are impaired relative to the wildtype. We will measure DNA binding activity of hFEN1 mutants using gel shift with biotin‐labeled DNA substrate to validate if the results we observe are due to defects in DNA substrate binding.This work is funded by NIH R01CA076734: Fen1 in genome stability and cancer.