DNA polymerase ζ generates tandem mutations in immunoglobulin variable regions

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Low-fidelity DNA polymerases are crucial for introducing nucleotide substitutions during somatic hypermutation in immunoglobulin variable regions.
• DNA polymerase eta (pol η) is recognized as the primary low-fidelity polymerase, but the roles of other polymerases, like DNA polymerase zeta (pol ζ), remain unclear.
• Previous studies have yielded contradictory results regarding the involvement of pol ζ in somatic hypermutation.

Purpose of the Study:

• To investigate the potential contribution of pol ζ to somatic hypermutation, especially in the absence of pol η.
• To determine if pol η masks the activity of pol ζ.
• To characterize the mutational spectra and identify unique mutational signatures associated with pol ζ activity.

Main Methods:

• Generation of mice deficient for pol η and heterozygous for pol ζ (Polζ(+/-) Polη(-/-)).
• Analysis of hypermutation frequency and spectra in Polζ(+/-) Polη(-/-) clones compared to Polζ(+/+) Polη(-/-) controls.
• Examination of tandem double-base substitutions and contiguous mutations.
• Comparison of mutation data across various genetic backgrounds, including wild type and deficiencies in Ung, Msh2, and Msh6.

Main Results:

• No significant alteration in overall hypermutation frequency or spectra was observed between Polζ(+/-) Polη(-/-) and Polζ(+/+) Polη(-/-) clones.
• A notable decrease in tandem double-base substitutions was found in Polζ(+/-) Polη(-/-) cells compared to Polζ(+/+) Polη(-/-) cells.
• The unique signature of tandem mutations suggests pol ζ's capacity to extend a mismatch with a second mutation in vivo.
• Data indicate a potential functional link between pol ζ and the MSH2-MSH6 mismatch repair pathway.

Conclusions:

• Pol ζ actively contributes to generating tandem double-base substitutions during somatic hypermutation, a role masked by pol η.
• The unique mutational signature provides in vivo evidence for pol ζ's involvement in mutational synthesis.
• Pol ζ may operate within the MSH2-MSH6 DNA repair pathway, highlighting its complex role in immunoglobulin diversification.