Mass spectrometric investigation of the role of the linking polypeptide chain in DNA polymerase I.

DNA polymerase I offers great promise for a wide range of biotechnological applications due to its capability to add labeled nucleotides into double-stranded large DNA molecules by using both polymerase and nuclease domains. Accordingly, it is crucially important to thoroughly characterize this enzyme for further developments. Although the enzyme has been thus far characterized using mainly traditional analytical instruments, here we utilized an advanced and convenient means of mass spectrometry to elucidate enzymatic functions and mechanisms by measuring DNA oligomers generated by polymerase and nuclease reactions. Our analysis revealed several novel enzymatic features, including the observation that polymerase readily dissociates from the DNA molecules containing a wide single-stranded section. From this finding, we reasoned a serious situation of DNA break because polymerase domains cannot efficiently repair the wide single-stranded section, which is susceptible to DNA breaks. Furthermore, we deduced a plausible explanation for a paradoxical question as to why two domains of polymerase and 5'-nuclease are linked by a small and flexible polypeptide in polymerase I. The polypeptide link seems to prevent a 5'-nuclease from causing DNA breaks by locating a polymerase domain closely for immediate repair reaction. Here we present experimental evidence to prove our hypothesis via a set of mass spectrometric analyses as well as single DNA molecule observation and bacterial cell growth assay. Consequently, mass spectrometric analysis for DNA polymerase I provides a meaningful biological insight that a polypeptide link can be a molecular leash to control an aggressive domain in order to prevent unmanageable damages.

Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ-like exonuclease domain-containing protein with homohexameric assembly.

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ-like 3'-5' exonuclease domain-containing protein (AtDECP). The DnaQ-like 3'-5' exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed ß-sheet flanked by α-helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring-shaped structure and comparison with WRN-exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N-terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ-like 3'-5' exonuclease and its substrate nucleic acids.