Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA polymerase β activity☆
Graphical abstract
Introduction
DNA stability (t1/2 = 30 million years) inside cells is undermined by susceptibility to damage by various agents such as nucleophiles (free radicals) or through malfunctioning of DNA-binding enzymes (e.g. polymerases, ligases or topoisomerases) [1]. Cellular DNA experiences various insults during a cell's lifespan resulting in DNA damage (at about 7 lesions/cell/minute) [1]. Physical, chemical and/or biological insults result in a number (about fifteen different types) of observed DNA damage including fragmentation, deletion, nucleotide or base substitution [1]. The consequences of such damage lead to defective or inaccurate gene replication resulting in abnormal cell function and disease [1]. If not repaired, DNA damage leads to either cell death (e.g. degenerative neurological disorders) or mutations causing uncontrolled cell proliferation (as seen in cancer) [1]. The DNA repair machinery is a mechanism utilized by the cell to correct DNA damage. This repair machinery consists of different types of repair such as base excision repair, single and double-strand break repair [1]. Each type of repair consists of several key proteins performing correction function. DNA repair proteins are potential targets for therapeutic interventions [2]. DNA polymerase β is a key component of Base Excision Repair (BER). The enzyme performs the distributive polymerase gap-filling function as well as deoxyribose phosphate (dRP) lyase activity [3].
DNA polymerase β is a 39 kDa enzyme that comprises two major domains, a 31 kDa domain responsible for the polymerase activity and an 8 kDa domain, which bind ss DNA and has the dRP lyase activity [3]. The atomic structure has previously been elucidated and several crystal structures have since been published for the enzyme and its binary and ternary complexes ([[4], [5], [6]]). Key features of DNA polymerase β′s structure suggest its organization in three major structural sub-domains: C (catalytic), D (DNA binding), N (nascent base pair binding) [4,5] (see Fig. 1). These three sub-domains correspond to the palm, thumb, and fingers sub-domains, which were observed for other polymerases such as HIV-1 reverse transcriptase [6]. Each of these three structural sub-domains serves a specific function with the palm serving as the platform that holds the DNA template-primer, whilst palm and fingers align template/primer towards polymerase active site. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at S44 and 55, resulting in loss of its polymerase enzymatic activity, but not its ability to bind ssDNA [7]. The enzyme was also observed to be phosphorylated in vivo in HeLa cells nuclei (Idriss, H, Kumar A and Wilson SH, unpublished observation), although this has not been conclusively reproduced. Several DNA polymerases are regulated through reversible post-translational modifications and DNA polymerase β was shown to be post-translationally phosphorylated, acetylated and methylated with functional consequences [[7], [8], [9], [10]].
Since PKC phosphorylation affects DNA Polymerase β′s polymerase activity, we argued that phosphorylation should also alter the enzyme's structure, as well as its overall charge. Therefore, we set out to simulate potential phosphorylation-induced structural changes for DNA polymerase beta using molecular modeling force field (CHARMM22) and published structural coordinates (pdb: 2FMS, 3ISB, and 1BPD). Other computational studies exist that looked at various aspects of the enzyme's structure/function [[11], [12], [13]].
Section snippets
Objectives
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Molecular dynamics simulation of DNA polymerase β in its apo state and its phosphorylated structure at S44, S55, and S44/55.
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Structural and dynamics study of DNA polymerase β and its phosphorylated structure via MD simulation.
Methodology
The X-ray crystal structures of DNA polymerase β (PDB entries: 2FMS [14], 3ISB [15], and 1BPD [16]) were downloaded from PDB. 2FMS is the ternary complex (human DNA polymerase β), 3ISB is the binary complex (human DNA polymerase β), and 1BPD is free DNA polymerase β (rat). Coordinates of the DNA substrate and dUMPNPP in 2FMS were removed to yield a structure which only contains the protein. Using this DNA polymerase β structure, separate structures with (1) S44 (pS44), (2) S55 (pS55), (3) S44
Result and discussion
DNA polymerase β assumes several intermediate structures along its reaction pathways [5]. The unliganded enzyme has an extended structure (1BPD, see Fig. 1A). It adopts a doughnut-like open structure after binding DNA (3ISB, see Fig. 1B) which undergoes further conformational changes to the closed structure after binding dNTP (2FMS, see Fig. 1C). In this work, we studied the effects of phosphorylation on the enzyme using MD simulations of several systems. The systems include five 2FMS systems:
Conclusion
Phosphorylation at S44 had induced major conformational fluctuation from apo_2FMS at the single strand DNA binding domain and dNTP selection domain. Adding a negative charge to S44 probably disrupts interactions between the lyase domain and the N-sub-domain of the polymerase (S44 and E335 H-bond in 2FMs). These conformation changes destabilize the closed structure. Thus the binding of single-strand DNA or dNTP might be affected by phosphorylation at these specific residues, causing DNA
Acknowledgement
This project is a personal and voluntary initiative by Dr. Haitham Idriss in support of Palestinian science [30]. We thank Dr. William (Bill) A. Beard (NIEHS, NIH) for discussion and for supplying Fig. 4. NAMD simulations runs were performed in the 1200 cores HPC of Khalifa University. Dr. Haitham Idriss dedicates this work to the memory of his late brother Mr. Hisham Idriss (1952–2012). Dr. Haitham Idriss is grateful for his mother, Hajjah Mrs. Zahia Idriss (1929-2018), for her generous
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This article first appeared in volume 79, please see publisher's note: https://doi.org/10.1016/j.jmgm.2017.11.002.
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Annals of AlQudsMedicine (www.annalqudsmed.com).