DNA replication represents a series of precisely regulated events performed by a complex protein machinery that guarantees accurate duplication of the genetic information. Since DNA replication is permanently faced by a variety of exogenous and endogenous stressors, DNA damage response, repair and replication must be closely coordinated to maintain genomic integrity. HROB has been identified recently as a binding partner and activator of the Mcm8/9 helicase involved in DNA interstrand crosslink (ICL) repair. We identified HROB independently as a nuclear protein whose expression is co-regulated with various DNA replication factors. Accordingly, the HROB protein level showed a maximum in S phase and a downregulation in quiescence. Structural prediction and homology searches revealed that HROB is a largely intrinsically disordered protein bearing a helix-rich region and a canonical oligonucleotide/oligosaccharide-binding-fold motif that originated early in eukaryotic evolution. Employing a flow cytometry Förster resonance energy transfer (FRET) assay, we detected associations between HROB and proteins of the DNA replication machinery. Moreover, ectopic expression of HROB protein led to an almost complete shutdown of DNA replication. The available data imply a function for HROB during DNA replication across barriers such as ICLs.
Keywords: DNA interstrand crosslink (ICL) repair; DNA replication; Förster resonance energy transfer (FRET); HROB; protein overexpression; soluble protein fragment identification.