Okazaki fragment maturation: DNA flap dynamics for cell proliferation and survival

Abstract

Unsuccessful processing of Okazaki fragments leads to the accumulation of DNA breaks which are associated with many human diseases including cancer and neurodegenerative disorders. Recently, Okazaki fragment maturation (OFM) has received renewed attention regarding how unprocessed Okazaki fragments are sensed and repaired, and how inappropriate OFM impacts on genome stability and cell viability, especially in cancer cells. We provide an overview of the highly efficient and faithful canonical OFM pathways and their regulation of genomic integrity and cell survival. We also discuss how cells induce alternative error-prone OFM processes to promote cell survival in response to environmental stresses. Such stress-induced OFM processes may be important mechanisms driving mutagenesis, cellular evolution, and resistance to radio/chemotherapy and targeted therapeutics in human cancers.

Keywords: DNA damage response; RNA primer; synthetic lethality; tandem duplication.

Figure 1.. Two canonical OFM processes via formation and cleavage of short or long 5’ flaps and nick ligation.

Pol δ-mediated Okazaki fragment extension encounters an RNA–DNA primer in a downstream Okazaki fragment and displaces the primer portion to form a 5′ flap. In most cases, the 5′ flap is 2–10 nt in length (short flap) and is primarily cleaved by FEN1, creating a DNA nick for ligation. EXO1 also degrades a relatively small percentage of such short 5′ flaps and serves as a backup of FEN1. The displacement-cleavage cycle continues until the whole RNA–DNA primer is degraded, which also removes any Pol α errors (dark dots in the RNA–DNA primer). The created ligatable DNA nick is then ligated by Lig I. This process is called short flap OFM. If a short RNA–DNA flap escapes cleavage by FEN1 or EXO1, continuous DNA strand displacement may result in a long 5′ flap of up to 300 nt in length. On the other hand, if PIF1 helicase is recruited to the replisome, DNA strand displacement is enhanced, leading to the formation of a long 5′ flap. The ssDNA binding protein RPA binds to the long RNA–DNA flap, inhibiting FEN1 cleavage of the 5′ flap. However, RPA recruits DNA2, which cleaves the long 5′ flap in the middle and creates a short 5′ flap (less than 10 nt). The short flap is then cleaved by FEN1 or EXO1, and the DNA nick is ligated by Lig I. The process that removes the long 5′ flap by the sequential actions of DNA2 and FEN1 (or EXO1) is called long flap OFM. In both short flap and long flap OFM, if the Pol α errors are not removed by 5′ flap cleavage, they can be edited out by the exonuclease activity of FEN1.

Figure 2.. Sequential post-translational modifications of FEN1 mediate its dynamic interactions with PCNA and timely exchange of Okazaki fragment enzymes.

During lagging strand DNA replication in S phase, FEN1 is symmetrically methylated at arginine residues, primarily R192 (R192me2s FEN1). The R192 methylation of FEN1 promotes its interaction with PCNA for recruitment to 5′ flaps. Once methylated FEN1 completes its RNA–DNA primer removal function, it is demethylated by an arginine demethylase, typified by JMJD1B. R192 demethylation of FEN1 allows its binding to CDK1, which catalyzes S187 phosphorylation of FEN1. S187 phosphorylates FEN1, dissociates from PCNA and undergoes sequential type 3 SUMO modification (K168) and ubiquitination (K354), leading to FEN1 degradation. Dissociation from PCNA as well as degradation of FEN1 ensure the efficient binding of Lig I to PCNA for DNA ligation.

Figure 3.. DNA lesions, damage responses, and cellular consequences of OFM defects.

Genetic and epigenetic alterations and various environmental insults can give rise to OFM defects or failure, which can result in ssDNA nicks or gaps with or without a short or long 5′ flap and ssDNA gaps with a secondary structure such as G4 opposite to the gap. DNA nicks or gaps can be further converted into DSBs by endonuclease cleavage or due to the collapse of replication forks in the next round of DNA replication. DNA damage sensor proteins, including RPA, the 9-1-1 complex, and PARP1 (in mammalian cells only), recognize these DNA lesions and activate the DNA damage response kinases ATM and ATR, which phosphorylate CHK2 and CHK1, respectively. Further downstream, these induce permanent damage, cellular senescence, and death or cell cycle arrest, DNA repair, and cell survival, respectively. Death or survival depends on the balance of ATM- or ATR-triggered pro-death and pro-survival signaling, and this balance hinges on the type and level of lesions in the cells. While SSBs are mostly not lethal, DSBs caused by fork collapse frequently lead to cell death. In mammalian cells, the cell fate decision made in response to various types and levels of DNA lesions is controlled by ATM/CHK2- or ATR/CHK1-mediated phosphorylation of the transcription factor p53, which induces the expression of both pro-death and pro-survival genes. Distinct p53 phosphorylation statuses determine the balance among pro-death and pro-survival p53 target genes, leading to cell death or cell survival.

Figure 4.. Flap dynamics lead to a 3′ flap for alternative OFM pathways and cell survival.

Under normal physiological conditions, unprocessed 5′ flaps may anneal to the template strand, creating DNA nicks for ligation and classic duplications. Under conditions of stress, cells activate additional DNA damage response pathways, leading to transformation of 5′ flaps into 3′ flaps. Nucleolytic degradation of 3′ flaps results in DNA nicks for Okazaki fragment ligation, called 3′ flap OFM. In 3′ flap OFM, Pol α-introduced errors are not removed, potentially causing base substitutions. Meanwhile, in certain regions, 3′ flaps may form a fold-back structure or anneal to nearby regions of microhomology. Extension and ligation of such 3′ flaps result in alternative duplications. Alternatively, the 3′ flap may invade the sister chromatin and initiate the template switching process for OFM. This process requires the involvement of HDR proteins and leads to sister chromatin exchange.

Figure 5.. The template switching process facilitates OFM in difficult-to-replicate regions.

Stable secondary structures such as G4 frequently form in difficult-to-replicate regions, including telomeres and centromeres, blocking Pol δ-driven Okazaki fragment extension and OFM and resulting in a gap on the nascent strand, with a secondary structure (G4) on the template strand. During or after DNA replication, cells utilize the template switching process to repair gaps and bypass stable secondary structures. This process includes sequential nuclease-mediated gap resection, helicase (PIF1)-mediated duplex unwinding to create a 3′ flap, and 3′ flap invasion into the sister chromatin. This process creates a Holliday junction (HJ) structure, which requires HJ resolvase to resolve. The bypassed G4 structure in the genome may be resolved by DNA2-mediated G4 cleavage or removed by nucleotide excision repair.

Figure 6.. OFM inhibition creates multiple layers of synthetic lethality (SL) with PARP1 inhibition or BRCA1/2 deficiency in cancer cells.

Inhibition of OFM enzymes such as RNasH2, FEN1, DNA2, or EXO1 in human cancer cells leads to the formation of un-ligated Okazaki fragments, which recruit PARP1 for SSB repair. Inhibition of OFM and PARP1 synergistically drives the accumulation of SSBs that can be converted into DSBs and/or stalled/collapsed replication forks. DSB repair at least partly requires the BRCA1/2-mediated HDR pathway. Thus, OFM inhibition creates SL with BRCA1/2 deficiency. In addition, OFM enzymes, including DNA2 and EXO1, work together with other nucleases including CtIP and MRE11 to resect DNA end for DSB repair via the HDR pathway. Thus, the inhibition of OFM enzymes may impair HDR as well, contributing to SL with PARP1 inhibitors.