Replication stress as a driver of cellular senescence and aging.

Replication stress refers to slowing or stalling of replication fork progression during DNA synthesis that disrupts faithful copying of the genome. While long considered a nexus for DNA damage, the role of replication stress in aging is under-appreciated. The consequential role of replication stress in promotion of organismal aging phenotypes is evidenced by an extensive list of hereditary accelerated aging disorders marked by molecular defects in factors that promote replication fork progression and operate uniquely in the replication stress response. Additionally, recent studies have revealed cellular pathways and phenotypes elicited by replication stress that align with designated hallmarks of aging. Here we review recent advances demonstrating the role of replication stress as an ultimate driver of cellular senescence and aging. We discuss clinical implications of the intriguing links between cellular senescence and aging including application of senotherapeutic approaches in the context of replication stress.

Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.