Effective transcription, replication, and maintenance of the genome require a diverse set of molecular machines to perform the many chemical transactions that constitute these processes. Many of these machines use single-stranded nucleic acids as templates, and their actions are often regulated by the participation of nucleic acids in multimeric structures and macromolecular assemblies that restrict access to chemical information. Superfamily II (SF2) DNA helicases and translocases are a group of molecular machines that remodel nucleic acid lattices and enable essential cellular processes to use the information stored in the duplex DNA of the packaged genome. Characteristic accessory domains associated with the subgroups of the superfamily direct the activity of the common motor core and expand the repertoire of activities and substrates available to SF2 DNA helicases, translocases, and large multiprotein complexes containing SF2 motors. In recent years, single-molecule studies have contributed extensively to the characterization of this ubiquitous and essential class of enzymes.

DNA polymerases use either a bulky active site residue or a backbone segment to select against ribonucleotides in order to faithfully replicate cellular genomes. Here, we demonstrated that an active site mutation (Y12A) within Sulfolobus solfataricus DNA polymerase IV (Dpo4) caused an average increase of 220-fold in matched ribonucleotide incorporation efficiency and an average decrease of 9-fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000-fold in sugar selectivity. Thus, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation. Other than synthesizing DNA as the wild-type Dpo4, the Y12A Dpo4 mutant incorporated more than 20 consecutive ribonucleotides into primer/template (DNA/DNA) duplexes, suggesting that this mutant protein possesses both a DNA-dependent DNA polymerase activity and a DNA-dependent RNA polymerase activity. Moreover, the binary and ternary crystal structures of Dpo4 have revealed that this DNA lesion bypass polymerase can bind up to eight base pairs of double-stranded DNA which is entirely in B-type. Thus, the DNA binding cleft of Dpo4 is flexible and can accommodate both A- and B-type oligodeoxyribonucleotide duplexes as well as damaged DNA.