Human DNA polymerase η has reverse transcriptase activity in cellular environments.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Classical DNA and RNA polymerases have distinct roles, but some exhibit multiple functions.
• Human DNA polymerase eta (hpol η) was previously shown to incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) using DNA and RNA substrates.
• Structural studies identified key residues (Phe-18, Tyr-92) influencing sugar selectivity in hpol η.

Purpose of the Study:

• To investigate the physiological relevance of hpol η's ability to incorporate rNTPs into DNA.
• To determine if hpol η functions as a reverse transcriptase in human cells under physiological conditions.
• To assess the role of hpol η in DNA synthesis using RNA primers.

Main Methods:

• Biochemical assays using purified hpol η and cell extracts.
• Experiments involving XP-V fibroblast cell lines lacking hpol η and HEK293T cells with manipulated hpol η expression.
• Analysis of rNTP incorporation opposite DNA lesions and subsequent substrate cleavage by RNase H2.
• Comparison with other translesion synthesis (TLS) polymerases (hpol ι, κ, ζ).

Main Results:

• Purified hpol η incorporates rNTPs into DNA primers at physiological concentrations, even with competing dNTPs.
• The presence of incorporated rNTPs reduced substrate cleavage by RNase H2.
• XP-V cells lacking hpol η showed impaired rNTP incorporation into DNA, which was restored by hpol η expression.
• HEK293T cells demonstrated hpol η-dependent dNTP incorporation into RNA-primed DNA, a function lost upon hpol η deletion.
• Other TLS polymerases did not exhibit similar functional redundancy when individually deleted.

Conclusions:

• Human DNA polymerase eta (hpol η) possesses significant reverse transcriptase activity in vivo.
• hpol η plays a major role in physiological processes requiring the incorporation of ribonucleotides into DNA.
• These findings highlight the versatile functions of hpol η beyond its canonical DNA polymerase activity.