Ribonucleotides and manganese ions improve non-homologous end joining by human Polμ

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Human DNA polymerase mu (Polμ), a DNA repair enzyme, exhibits both template-directed and template-independent (terminal transferase) activities.
• Polμ, similar to terminal deoxynucleotidyl transferase (TdT), can incorporate ribonucleotides (NTPs) instead of deoxynucleotides (dNTPs), though the physiological role remains unclear.

Purpose of the Study:

• To investigate the role of NTPs in Polμ-mediated non-homologous end joining (NHEJ).
• To determine the effect of manganese ions (Mn2+) on Polμ activity during NHEJ.
• To explore the structural basis for Polμ's substrate versatility.

Main Methods:

• Enzymatic assays using purified human Polμ.
• Analysis of Polμ activity with varying nucleotide and metal ion concentrations.
• Site-directed mutagenesis of the Polμ active site, focusing on the 'steric gate'.

Main Results:

• Polμ efficiently incorporated NTPs instead of dNTPs during the NHEJ of non-complementary DNA ends.
• Physiological concentrations of Mn2+ enhanced both the efficiency and accuracy of Polμ-mediated NHEJ.
• Mutational analysis revealed that an open active site in Polμ accommodates alternative nucleotides and metal ions.

Conclusions:

• Polμ's ability to utilize NTPs and its adaptability to different metal ions contribute to its versatile role in DNA repair.
• This substrate flexibility allows Polμ to optimize nucleotide selection for individual NHEJ events, enhancing efficiency without compromising fidelity.
• The findings expand our understanding of DNA repair mechanisms and the functional plasticity of DNA polymerases.