Structural Biochemistry/Nucleic Acid/DNA/Replication Process/DNA Initiation

DNA initiation is the first stage of the DNA replication process. During this stage, the double stranded DNA (dsDNA) is first separated into single strands by breaking up the hydrogen bonds between base pairs. The separation of dsDNA into singled stranded DNA (ssDNA) is known as DNA melting. Proteins that are responsible for breaking up of dsDNA are called initiator proteins. In the next step, proteins called helicase bind to the dsDNA and unwind it to create a replication fork. In Eukaryotic and archaeal cells, melting and unwinding of DNA are mainly accomplished by mini-chromosome maintenance helicase (MGM) along with multiple initiation proteins. However, helicase such as large T antigen (LTag) and E1 which are found in simian virus 40 (SV40) and bovine paillomavirus (BPV) are able to break up and unwind the dsDNA without any additional cofactors (Chen et al.). Because of the great similarity between the viral and eukaryotic and archaeal DNA replication system, LTag and E1 are studied intensively by researchers in hope of gaining a better understanding in replication process.

Mechanism
Differences in the arrangement of β-hairpins and mode of ATP binding etc. in the viral proteins can lead to different mechanism of melting and unwinding. For example, LTag of SV40 is believed to use mechanism that follows the double-pump looping model which is described in Chen et al. First, LTag in the shape of a double-hexamer binds to the dsDNA at the replication origin and compress it to break up the hydrogen forces between two strands. Two hexamers ahead of the replication origin then pump the dsDNA into the double-hexamer to create a replication fork that is consisted of ssDNA as loops. Further pumping of the dsDNA will elongate the replication fork to allow fork progression. On the other hand, E1 of BPV uses mechanism that follows closely with the steric exclusion model (Chen et al.). In this model, E helicase exists only as a single hexamer and is separated into two trimers with each binds to one strand of dsDNA at the origin to induce melting. After successfully breaking up the dsDNA, two trimers rejoin to form a hexamer that binds to only one strand of dsDNA and unwinds it to create a replication fork.

Conclusion
Although they present plausible mechanisms for DNA melting and formation of replication fork, both models still require support of further evidences. Questions such as how LTag binds to dsDNA or whether the E1 hexamer can separate into two trimers still remain unanswered. More intensive investigation and research are therefore needed.

Reference
Chen, Xiaojiang S, Paul Chang and Dahai Gai. "Origin DNA melting and unwinding in DNA replication". Current Opinion in Structural Biology 2010, 20:1-7.