Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

Abstract

During S-phase, minor DNA damage may be overcome by DNA damage tolerance (DDT) pathways that bypass such obstacles, postponing repair of the offending damage to complete the cell cycle and maintain cell survival. In translesion DNA synthesis (TLS), specialized DNA polymerases replicate the damaged DNA, allowing stringent DNA synthesis by a replicative polymerase to resume beyond the offending damage. Dysregulation of this DDT pathway in human cells leads to increased mutation rates that may contribute to the onset of cancer. Furthermore, TLS affords human cancer cells the ability to counteract chemotherapeutic agents that elicit cell death by damaging DNA in actively replicating cells. Currently, it is unclear how this critical pathway unfolds, in particular, where and when TLS occurs on each template strand. Given the semidiscontinuous nature of DNA replication, it is likely that TLS on the leading and lagging strand templates is unique for each strand. Since the discovery of DDT in the late 1960s, most studies on TLS in eukaryotes have focused on DNA lesions resulting from ultraviolet (UV) radiation exposure. In this review, we revisit these and other related studies to dissect the step-by-step intricacies of this complex process, provide our current understanding of TLS on leading and lagging strand templates, and propose testable hypotheses to gain further insights.

Figure 1

Semi-discontinuous DNA replication from a replication bubble. Parental and nascent (daughter) DNAs are shown in black and blue, respectively, with arrows indicating the 5′ to 3′ direction of DNA synthesis. At each origin of replication (Ori, shown in green), two replication forks are established and progress in opposite directions (indicated by grey arrows) as the two template strands of each fork are replicated in concert. The leading strand templates are replicated continuously (solid line) in the direction of replication fork progression while the nascent DNA on the lagging strand templates is synthesized discontinuously (dashed line) in the opposite direction.

Figure 2

A model of the eukaryotic replisome assembled at each replication fork. This model shown in cartoon form was generated from recent EM structures^, and related studies (cited in main text). This model suggests that leading strand synthesis and at least the initiation of an Okazaki fragment occur on opposite sides of the helicase, necessitating an unexpected path for the templates as the dsDNA is unwound. The lagging strand template is sterically occluded from the central chamber of the MCM core and traverses the outside of the MCM core to reach pol α-primase. The leading strand template enters the CTD tier of the MCM core, traverses the central chamber or exits at an internal position, and then bends upward toward pol ε.

Figure 3

The most prominent DNA lesions resulting from exposure to UV radiation. The numerical positions within the six-membered rings are indicated in blue. Exposure of adjacent pyrimidines, such as two thymidines, results in formation of (6–4) PPs and CPDs. The proportion of each is indicated. The only mechanism for repairing these photodimers within the human genome is the NER pathway, which proceeds by two sub-pathways: GG-NER and TC-NER. (6–4) PPs are efficiently and rapidly repaired by GG-NER while CPDs are repaired via TC-NER in a much slower fashion.

Figure 4

Pol α-primase encountering a UV-induced lesion ( ) on a lagging strand template. This model shown in cartoon form only displays pol α-primase on the lagging strand template for simplicity.

Figure 5

Pol ε encountering a UV-induced lesion within a leading strand template. This model shown in cartoon form depicts discontinuous lagging strand DNA synthesis as a dashed line for simplicity. Arrows indicate the direction of DNA synthesis (5′ ➜ 3′). The heterohexameric MCM core is depicted as a barrel. (1) Upon encountering a UV-induced lesion ( ) within a leading strand template, DNA synthesis by pol ε abruptly stops but the CMG helicase remains intact and continues unwinding DNA, exposing long stretches of the leading strand template. During such uncoupling, RPA coats the exposed leading strand template and DNA synthesis continues on the undamaged lagging strand template as the replication fork progresses. Furthermore, non-replicating pol ε maintains contact with the CMG helicase and is carried downstream of the offending lesion while PCNA is left behind at the blocked P/T junction. Pol ε may also maintain contact with the blocked P/T junction upon uncoupling, forming ssDNA loop. However, such complexes are expected to be short-lived due to RPA●ssDNA interactions. (2) Uncoupled from leading strand DNA synthesis, the CMG helicase and, hence, lagging strand DNA synthesis eventually stall downstream of the offending damage, halting progression of the replication fork.

Figure 6

Pol δ encountering a UV-induced lesion within a lagging strand template. For simplicity, this model shown in cartoon form depicts continuous leading strand DNA synthesis as a solid line and only displays PCNA, RPA and pol δ proteins on the lagging strand template. Arrows indicate the direction of DNA synthesis (5′ ➜ 3′). Upon encountering a UV-induced lesion (X) within a lagging strand template, pol δ rapidly and passively dissociates into solution, leaving PCNA behind on the DNA. Pol δ may reiteratively dissociate and re-bind to the PCNA encircling the blocked P/T junction but pol δ-mediated DNA synthesis will not resume on the afflicted Okazaki fragment until the lesion is bypassed. Meanwhile, pol ε continues to replicate the undamaged leading strand template as the CMG helicase unwinds the duplex DNA. In this schematic, “scheduled” re-priming of the lagging strand template by pol α-primase has not yet occurred.

Figure 7

“On the fly” TLS on a leading strand template. An unrepaired UV-induced lesion ( ) encountered in a leading strand template leads to stalling of the replication fork downstream of the lesion (as depicted in Figure 5). TLS on a leading strand template can occur by one of two pathways. In “on the fly” TLS, bypass of the UV-induced lesion occurs before progression of the replication fork has been re-started through a re-priming event, as follows: 1) One or more TLS pols bypass the offending DNA lesion, extending the aborted primer terminus to an undamaged section of the leading strand template. 2) Pol δ faithfully extends the primer to the stalled CMG helicase where 3) the bound pol ε rapidly replaces pol δ on the leading strand template, re-starting progression of the stalled replication fork. In this scenario, replication fork re-start requires TLS. As an alternative to “on the fly” TLS, UV-induced lesions within a leading strand template may be bypassed by postreplicative gap-filling (Figure 8).

Figure 8

Postreplicative gap-filling on a leading strand template. An unrepaired UV-induced lesion ( ) encountered in a leading strand template leads to stalling of the replication fork downstream of the lesion (as depicted in Figure 5). In postreplicative gap-filling, bypass of the UV-induced lesion occurs after the replication fork has been re-started through a re-priming event, as follows. 1) PrimPol is recruited to the RPA-coated ssDNA downstream of a UV-induced lesion by a direct interaction with RPA. Once localized, PrimPol synthesizes a nascent primer at a random location downstream of the offending damage and a new PCNA ring is loaded onto the nascent 3′ hydroxyl terminus. This leaves behind an RPA-coated ssDNA gap (postreplicative gap) containing PCNA and the lesion. 2) Pol δ faithfully extends the nascent primer terminus to the stalled CMG helicase where 3) the bound pol ε rapidly replaces pol δ on the leading strand template, re-starting progression of the stalled replication fork. 4) Eventually, the offending DNA lesion within the postreplicative gap (shown) is bypassed by one or more TLS pols, extending the aborted primer terminus to an undamaged section of the leading strand template. 5) Pol δ “fills in” the remainder of the postreplicative gap and the 5′ RNA end of the downstream duplex region is removed as in Okazaki fragment processing/metabolism. 6) The resident PCNA is removed some time after the fully-extended primer terminus is ligated to the 5′ end of the downstream daughter DNA.

Figure 9

Postreplicative gap-filling on a lagging strand template. DNA synthesis by pol δ abruptly stops upon encountering a UV-induced lesion ( ) within a lagging strand template but the replisome and, hence, the replication fork continue unimpeded (as detailed in Figure 6). Bypass of the offending damage occurs by postreplicative gap-filling as follows. 1) Pol α-primase performs “scheduled” synthesis of an RNA/DNA hybrid primer upstream (5′) of the offending damage on the exposed lagging strand template. PCNA residing at the blocked P/T junction remains and a pol δ holoenzyme is assembled on the nascent P/T junction with a new PCNA ring, allowing lagging strand DNA synthesis to resume and continue upstream of the offending DNA lesion. This prevents excessive exposure of the lagging strand template and generates a postreplicative gap less than or equal to the size of an Okazaki fragment (~100 – 250 nt) extending from the blocked P/T junction to the 5′ terminus of the downstream Okazaki fragment. As the replisome progresses in the absence of TLS, the replication fork moves further and further ahead of the postreplicative gap. 2) Eventually, one or more TLS pols bypass the offending DNA lesion within the postreplicative gap, extending the aborted primer terminus to an undamaged section of the lagging strand template. 3) Pol δ then “fills in” the remainder of the postreplicative gap and the 5′ RNA end of the downstream duplex region is removed as in Okazaki fragment processing/metabolism. 4) The resident PCNA is removed some time after the fully-extended primer terminus is ligated to the 5′ end of the downstream Okazaki fragment.

Figure 10

Dynamics of PCNA on a UV-damaged template. During DDT, the retention of PCNA at a blocked P/T junction abutting a UV-induced lesion ( ) is governed by three activities; 1) Enzymatic loading of PCNA onto the P/T junction; 2) Enzymatic unloading of PCNA from the P/T junction and; 3) diffusion of PCNA along either the nascent dsDNA or the adjacent ssDNA. If PCNA can vacate a blocked P/T junction, limiting PCNA rings will not be continuously re-loaded. However, diffusion of PCNA along DNA and enzyme-catalyzed unloading PCNA from DNA are prohibited during TLS, promoting retention of PCNA at blocked P/T junctions during S-phase.

Figure 11

Accessibility of postreplicative gaps. (A) Chromatin dynamics during S-phase. On the lagging strand template, Okazaki fragments yet to be ligated are indicated by dashed lines. During S-phase, nucleosomes ahead of a replication fork are disassembled as the replication fork progresses. Immediately following passage of a replication fork, nucleosomes are re-formed on the nascent daughter DNA by various histone chaperones. B) Monoubiquitination of PCNA and CAF-1 binding. Surface of the human PCNA ring generated with VMD (PDB 1AXC). Coloring scheme is indicated. Each PCNA monomer consists of two independent domains joined by an interdomain connecting loop (IDCL). Three PCNA monomers arrange in a head-to-tail manner, resulting in a ring with structurally distinct faces. The “front” face contains the IDCLs and interacts with pols. Following UV irradiation, single ubiquitin moieties are covalently attached to PCNA rings at the conserved lysine residue 164 (K164). C) Ubiquitin docking site 2. Shown in cartoon form is a side profile of a PCNA subunit-subunit interface. Coloring scheme is identical to panel B. At the interface, the N-terminal domain from one PCNA monomer interacts with the C-terminal domain from the adjacent monomer. At ubiquitin docking site 2, a ubiquitin moiety conjugated to K164 interacts with loop J (indicated) and residues within the subunit-subunit interface. (D) Selective inhibition of CAF-1 activity at postreplicative gaps. Upon formation of postreplicative gaps within either template, chromatin assembly continues in the wake of a progressing replication fork. PCNA is retained at a postreplicative gap and is monoubiquitinated by Rad6/Rad18. We propose that the ubiquitin moieties dock onto the PCNA ring at ubiquitin docking site 2, sheltering/disrupting the CAF-1 binding sites on PCNA. This precludes CAF-1 binding to PCNA, selectively inhibiting nucleosome assembly at/near a postreplicative gap until the gap is filled.