by Disha Shantaram Shenai

DNA Polymerase theta and its reverse transcriptase activity

In a eukaryotic cell, the nucleus contains a long molecule called DNA or deoxyribonucleic acid. It is like an instruction manual which contains the information needed to make proteins in the body. In 1958, Sir Francis Crick, proposed a theory called the ‘Central Dogma’. It explains the flow of information from DNA to RNA to produce functional proteins.

Crick further stated that the DNA code was transcribed to RNA which acts like small portable messages. RNA travels to ribosomes where it is translated to make proteins. He also stated that existing DNA can make new DNA by the process of replication. The steps of DNA to protein (without RNA) and RNA to DNA (structurally feasible) had no evidence, but Crick hypothesized that they seemed possible.

However, on 11th June, an article published in ScienceAdvances hypothesizes that human DNA polymerase θ (Polθ) is capable of RNA-dependent DNA polymerization activity also known as Reverse Transcription.

What is reverse transcription?

What does Polθ do?

What makes it different?

Let me try and break it down for you according to my undergraduate level of understanding.

What is reverse transcription?

In 1970, David Baltimore and Howard Temin independently and simultaneously discovered the enzyme Reverse Transcriptase in retroviruses (then known as RNA tumor viruses). This new enzyme could synthesize cDNA (complementary DNA) from an RNA template (viral genome), which helped these viruses to replicate faster within the host cells which had DNA as their genetic material.

Viral propagation in Retroviruses

Other roles of reverse transcriptase:

Besides retroviral propagation, the enzyme reverse transcriptase is also involved in various processes, both in prokaryotes and eukaryotes:

DNA Polymerase θ (Polθ):

DNA polymerase are enzymes that catalyse the synthesis of DNA from its monomers i.e., nucleotide triphosphates. For this, the enzyme adds nucleotides to the 3’-end of the DNA strand at the hydroxyl group. Eukaryotes have at several DNA polymerase (14 in mammalian cells) widely grouped into 7 families depending on their structure, homology, and function.  

Also known as DNA polymerase Q (PolQ), Polθ is an A-family polymerase encoded by POLQ gene. It is known to have a polymerase domain at its C-terminal and a helicase-like domain at the N-terminal. It plays multiple roles in DNA repair and contributes to genome stability.

Functions:

  • It enables MMEJ (microhomology-mediated end joining) repair pathway, which is an alternative to NHEJ (non-homologous end joining) in response to double stranded breaks in DNA. This pathway is highly error prone (due to inactive proof-reading domain) and may lead to mutations by deletions from the strand being repaired.
  • It inhibits homologous recombination repair mechanism.
  • Polθ uses many templates for its polymerase activity, i.e., DNA/DNA, DNA/RNA, single stranded and partial single stranded DNA. It shows low-fidelity DNA synthesis (fidelity refers to accuracy of replication of a template).
  • It also exhibits translesion synthesis (replication past DNA lesions) and lyase activity.
  • It has several functions such as interstrand-cross-link repair, base excision repair and DNA end joining.
  • Despite all these functions, the physiological role of Polθ under normal growth conditions is unclear.

It is important to note that Polθ is not expressed in all tissues. However, high expression is observed in cancer cells. This may correlate to poor chance of recovery, higher chance of recurrence, etc. It also confers resistance to genotoxic chemotherapies as it promotes survival of cells with DNA damage. As a result, Polθ is a promising potential drug target for anti cancer agents.

So what’s new?

Mammalian DNA polymerase enzymes involved in DNA repair were not known to possess the property of DNA polymerization by reading RNA, like reverse transcriptase in retroviruses. In this study, it is suggested that the main function of Polθ may be reverse transcription.

The Kleenow fragment of bacterial DNA Pol I may show reverse transcriptase activity after inactivation of its 3’-5’ proofreading activity. Polθ itself has a poor proofreading activity due to inactive domain. This prompted the hypothesis that it could serve as a RNA dependent DNA polymerase. On comparing with the reverse transcriptase from HIV, Polθ was found to be more efficient at this job and produced fewer errors. In fact, its RNA-dependent DNA polymerase activity seemed to be more efficient than its DNA dependent DNA polymerase activity, indicating that it could be the main function of this enzyme in the cell.

Besides this, X-ray crystallography studies showed that Polθ shows immense structural plasticity and is able to mould itself to incorporate larger and bulkier RNA.

This study of Polθ paves way for future studies in its potential role for RNA mediated DNA repair and cancer cell proliferation.

References:

20th June 2021