Template strand scrunching during DNA gap repair synthesis by human polymerase lambda

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Family X polymerases, like DNA polymerase lambda (Pol lambda), are involved in DNA repair.
• Pol lambda can bind both ends of short DNA gaps, facilitating repair.
• The mechanism of template nucleotide binding in gaps longer than 1 nucleotide was previously unknown.

Purpose of the Study:

• To elucidate the mechanism of DNA polymerase lambda (Pol lambda) in filling 2-nucleotide (nt) DNA gaps.
• To determine the structural basis for Pol lambda's interaction with longer DNA gaps.
• To investigate the role of conserved amino acids in Pol lambda's gap-filling activity and its impact on DNA repair.

Main Methods:

• X-ray crystallography to determine the structure of Pol lambda bound to a 2-nt DNA gap.
• Site-directed mutagenesis to replace conserved amino acids in the Pol lambda binding pocket.
• Biochemical assays to assess gap-filling processivity and non-homologous end joining (NHEJ) efficiency.

Main Results:

• Crystal structures revealed that Pol lambda "scrunches" the template strand in 2-nt gaps.
• An additional uncopied template base is bound in an extrahelical position within a specific binding pocket.
• Mutating conserved amino acids in this pocket reduced gap-filling processivity and NHEJ efficiency.

Conclusions:

• DNA polymerase lambda (Pol lambda) employs a template-scrunching mechanism for synthesizing DNA in 2-nt gaps.
• A conserved binding pocket with three amino acids is critical for this scrunching mechanism and efficient DNA repair.
• This scrunching mechanism during gap filling is analogous to that observed in RNA polymerase during transcription initiation.