ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• G-quadruplex (GQ) structures are formed by guanine-rich sequences and play roles in various cellular processes.
• Helicases and single-stranded DNA (ssDNA) binding proteins are known to unfold these structures, but the mechanisms are not fully understood.

Purpose of the Study:

• To investigate the kinetic mechanisms of Bloom helicase (BLM) interacting with human telomeric G-quadruplex (GQ) structures.
• To elucidate how BLM unfolds GQ structures and the factors influencing its activity and processivity.

Main Methods:

• Utilized single-molecule Förster resonance energy transfer (smFRET) to study BLM-DNA interactions.
• Employed partial duplex DNA (pdDNA) constructs with varying 5' ssDNA overhangs, some containing GQ structures.
• Performed kinetic studies under physiological salt and pH conditions.

Main Results:

• BLM localizes to the ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner.
• GQ unfolding was observed in 50-70% of BLM reeling attempts, with unsuccessful attempts leading to BLM dissociation.
• BLM-mediated GQ unfolding was often followed by refolding, and BLM exhibited significantly lower processivity compared to the helicase Pif1.

Conclusions:

• BLM unfolds GQ structures through a reeling mechanism, but its activity is limited by dissociation and lower processivity.
• The findings suggest potential common mechanisms among helicases for minimizing stable GQ formation duration.
• Understanding BLM's GQ interaction provides insights into DNA repair and genome stability mechanisms.