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Proliferating Cell Nuclear Antigen Interactions with Translesion DNA Polymerases


Lars Matkin '12 Nickolas Tun '12

Contents:

I. Introduction

Proliferating Cell Nuclear Antigen (PCNA) is an important protein complex that assists in DNA synthesis and repair. It functions as a sliding clamp that binds directly to multiple proteins and polymerases through a conserved binding domain called the PCNA-interacting protein box (PIP-box). However, the three Polymerases associated with Translesion synthesis, Polymerase Iota, Polymerase Kappa, and Polymerase Eta, have a non-canonical binding domain.

II. Canonical PCNA Interactions

PCNA is a homotrimeric protein that surrounds DNA, binding to Polymerase and anchoring it to the RNA primer sequence . Each individual monomer is comprised of an N and C terminus domain, which are linked by an interdomain connecting loop (IDCL) . Interacting proteins bind to PCNA through the canonical Pip-box which is composed of an eight amino acid sequence(QXXhXXaa) . The three conserved residues positioned within this sequence include a Gln at position one (p1), a hydrophobic residue at position 4 (p4), and two aromatic residues at position 7 (p7) and 8 (p8). The Gln at p1 in the PIP box is inserted into a pocket within the PCNA monomer (the hydrophobic socket). The p4 to p8 residues form into a helical structure, known as a hydrophobic plug that places the residues at p4, p7 and p8 into a hydrophobic pocket (socket) within the PCNA monomer. A second series of interactions exist between the C-terminus of the binding protein and the IDCL of the PCNA domain. This is facilitated through antiparallel beta sheet formation with the IDCL . This conserved sequence is typical of all protein interactions with PCNA, except for interactions with the three polymerases associated with translesion synthesis.

III. Non-canonical interactions between PCNA and Translesion Polymerases


All three of the eukaryotic translesion polymerases have a similar eight amino acid non-canonical PIP boxes that interact with the Hydrophobic Socket of PCNA . For instance Polymerase Iota, the polymerase responsible for bypassing (6-4) photoproducts, binds using the non-canonical Pip-box (KGLIDYYL) . Similarly Pol Kappa, which is primarily responsible for bypassing benzo[a]pyrene diol epoxide-adducted guanines, has a PIP-Box composed of the eight amino acid sequence (KHTLDIFF) ; and Polymerase Eta, which incorporates two adenines opposite a thymidine-thymidine (T-T) cyclobutane pyrimidine dimer, has the following non-canonical PIP-box: (MQTLESFF) . 


These non-canonical PIP-Boxes have four conserved residues: hydrophobic residues at p4 and p8, an acidic residue at p5, and an aromatic residue at p7 (XXXhcXah) (Clustal analysis). 


The conserved acidic residue at p5 allows for ion pair formation between His-44 of PCNA with each of the translesion polymerases . This ion pair interaction seem to contribute largely to the affinity of the polymerases to PCNA because mutant Pol Eta with E705A substitution, which lacks the acidic residue, exhibited much lower binding affinity. The other residues of the Pip-box vary between polymerases and results in different binding affinities between the translesion polymerases and the β sliding clamp.


IV. Differential Binding of the Translesion Polymerases


 Hydrogen bonds between PCNA and the polymerases influence the binding affinity between the proteins. Pol Iota, which has the greatest binding affinity for PCNA of all the translesion polymerases, has only one hydrogen bond between Met-429 and Gly-127 . Its superior binding is the result of close fitting shape complementarity to PCNA binding site. This is due to Leu-428 (p8) of Pol Iota forming a hydrophobic interaction with the Leu-126 of PCNA, outside of the hydrophobic socket .  ,and a β-bend-like structure created by Lys-421 (p1) forming an intermolecular hydrogen bond with the Tyr-427 (p7); the aliphatic domain of the Lys 421 side chain closely interacts with the aromatic ring of Tyr-426 (p6); and the carbonyl oxygen of the Lys-420 residue also interacts with the amide nitrogen of Leu-423 (p3) through hydrogen bonding . This β-bend structure is a crucial contributor to Pol Iota shape complementarity to PCNA .

Pol Eta, which has hydrogen bonds between Leu-711 and Gln-125, Leu-704 and His-44, as well as Gly-700 and Ile-255, has the greatest number of H-bond interactions . Despite the number of H-bonds formed Pol Eta has weaker binding than Pol Iota because Met-701 (p1) is forced into the hydrophobic pocket , reducing its binding integrity .
 
Pol Kappa has two hydrogen bonds between the binding domain and PCNA, one between Lys-870 and Gly-127 and another between Leu-865 and Val-45 .Despite these interactions it has minimal binding affinity with PCNA because Lys-862 (p1) is sterically hindered from the Hydrophobic Socket due to its large size and charge .

V. Biological Implications of the Non-canonical Interactions of TLS Polymerases with PCNA

PCNA interacts with Pol Iota, Pol Kappa, and Pol Eta through non-canonical PIP boxes that are highly conserved among mammalian species. Pol Iota and Pol Eta associate with PCNA stronger than Pol Kappa, due to their protein surface complementarities and hydrogen boding with PCNA. The protein surface complementarity, SC, of PCNA-Pol Iota and PCNA-Pol Eta are 0.78 and 0.75 respectively. Compared to the interaction of Pol Eta with PCNA, Pol Iota has fewer hydrogen bonds, but Pol Iota still maintains a comparably high affinity to PCNA due to its high SC value. The low affinity of Pol Kappa to the PCNA binding site is the result of SC value of 0.66 indicating poor complementary protein surface contact. These differential binding affinities explains why Pol Eta and Iota are more frequently recruited to DNA damage sites than Pol Kappa. Since these Polymerases are responsible for UV damage repair, their increased affinity for PCNA correlates with elevated recruitment during DNA replication.  Research has shown that cultured human cells suffer from increased UV sensitivity and tumor growth in the absence of Pol Eta and Iota. The infrequency of bulky adducts means that Pol Kappa is less commonly recruited than its UV damage repairing counterparts.

TLS polymerases have low processivity and fidelity because they are only required to overcome short damaged DNA sequences, after which replicative polymerases replace them. As DNA damage is detected, PCNA is monoubiquitinated by the RAD6-Rad18 complex through a hydrogen bond between Leu-8 of PCNA and Lys-110 of Ubiquitine . Once ubiquitination has occured translesion synthesis polymerases are subsequently recruited. How this signal is processed is unclear, however, ubiquitination of PCNA is a current area of study.

VI. References

Bruning J.B. & Shamoo Y. (2004) Structural and thermodynamic analysis of human PCNA with peptides derived from DNA polymerase-delta p66 subunit and flap endonuclease-1. Structure 12: 2209-2219

Gueranger Q., Stary A., Aoufouchi S. Faili A., Sarasin A., Reynaud C.A. & Weil J.C. (2008) Role of DNA polymerases ή, ί and ζ in UV resistance and UV-induced mutagenesis in a human cell line. DNA Repair 7: 1551-1562

Hishiki A., Hashimoto H., Hanafusa T., Kamei K., Ohashi E., Shimizu T., Ohmori H. & Sato M. (2009) Structural Basic for Novel Interactions between Human Translesion Synthesis  Polymerases and Poliferating Cell Nuclear Antigen. Journal of Biological Chemistry Vol 284, No. 16

Vidal A. E. & Woodgate R. (2009) Insights into the cellular role of enigmaticDNA polymerase ί. DNA Repair 8: 420-423

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