Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA

Abstract

PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)-a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations. SAXS | DNA replication | DNA repair | mutagenesis C lassical DNA polymerases-those involved in normal DNA replication and repair-cannot accommodate many types of DNA damage in the template strand. As a result, replication forks stall when encountering DNA damage, and this is one of the major sources of genome instability. One of the principal pathways for overcoming replication blocks is translesion synthesis (TLS) by one of a variety of specialized TLS polymerases (1-4). These TLS synthesis polymerases are recruited to stalled replication forks where they replace the classical polymerase and carry out DNA synthesis across from the DNA damage. DNA polymerase eta (pol η), for example, replicates efficiently and accurately through thymine dimers and 8-oxoguanine lesions (5, 6). Significant insights into how TLS polymerases accommodate DNA damage have come from structural and kinetic studies of these enzymes over the last decade (2, 4).Arguably the least understood steps of translesion synthesis are the recruitment of the TLS polymerase to stalled replication forks and the subsequent polymerase switch between the stalled classical polymerase and the TLS polymerase. These steps are governed by the monoubiquitylation of proliferating cell nuclear antigen (PCNA). PCNA is monoubiquitylated on Lys-164 by the Rad6-Rad18 complex in response to DNA damage (7,8). The at...

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

“…Structural data of monoubiquitinated PCNA and its conformations support the model that the secondary ubiquitin site allows the TLS polymerase Pol η to bind an occupied clamp and position it to compete for the cleft on transient dissociation of the replicative polymerase (42,43). In contrast to bacteria, where the occupancy of the rim site is controlled by polymerase concentration, analogous sites in eukaryotes are introduced by posttranslational modification.…”

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

“…The DNA was designed based on a crystallographic paper on human Fen1 (hFen1) in complex with DNA [13] and shown to be cleaved by Fen1 (Figure 4). The 'Okazakisome' was assembled from individually purified components, including a fully functional PCNA molecule in which the three subunits are expressed as a covalently-linked concatamer [2,6], which were loaded on GraFix gradients [8].…”

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

“…Therefore, only chain Y orientation was used in the model. The range of conformations observed in the crystal structure suggests segmental flexibility whereby a flexibly linked protein (such as FEN1) can adopt several discrete positions (30). To generate the initial model of the 9-1-1 ternary complex we used an overlay of the human 9-1-1 structure with truncated Rad9 subunit (9Δ-1-1; PDB ID code 3GGR) (20) onto the PCNA/FEN1/DNA model (Fig.…”