TY - JOUR

T1 - Structure and conformational dynamics of thehuman NEIL2 in complex with DNA

AU - Жданова, Полина

AU - Канажевская, Любовь Юрьевна

AU - Аксенова, Лилия Владимировна

AU - Коваль, Владимир Васильевич

AU - Chernonosov, Alexander A.

PY - 2023

Y1 - 2023

N2 - Living cells are protected from genotoxic damage by DNA repairenzymes. One of the repair pathways is base excision repair,BER. Enzymes in this pathway protect the genome from a var-iety of small non-volumetric lesions, which arise mainly fromdeamination, oxidation, etc. BER is initiated by excision of dam-aged bases, e.g. by DNA glycosylases. Human endonucleaseVIII-like protein 2 (hNEIL2) is a DNA glycosylase which prefersdamage in bubbles and other non-canonical DNA structures.Understanding the mechanisms of damage recognition andremoval, it is necessary to know the structural features of theenzyme. Among the attempts to study the structure of NEIL2proteins, only the opossum enzyme was crystallized and resolved.Using hydrogen deuterium exchange followed by mass spectro-metric analysis, we shown that in the free state the hNEIL2 pro-tein is in an open conformation. We also demonstrated that itcontains an extended disordered linker in the N-terminal domain(which makes crystallization nearly unfeasible). However, thestructure of the hNEIL2-DNA complex remains unclear. Weapplied a combination of HDX-MS and computer simulationmethods to study the structural and dynamic features of the for-mation of the human NEIL2 protein-substrate complex. Theexchange reaction was performed at 21°C and pH 7.6. The label-ing was terminated at time points between 10 s and 120 min byadding an aliquot of the reaction mixture to a quench buffer(0°C, pH 2.5) and immediately freezing the sample in liquidnitrogen. Immediately before analysis, the protein was thawedand subjected to cleavage on a column with immobilized pepsin.The peptides were retained on a trap column, concentrated anddesalted at 0°C, and then analyzed by MS. After processing thedata, we interpreted them by comparing them with the results ofcomputer simulations. This work was supported by grants fromthe RSF (20-14-00214).

AB - Living cells are protected from genotoxic damage by DNA repairenzymes. One of the repair pathways is base excision repair,BER. Enzymes in this pathway protect the genome from a var-iety of small non-volumetric lesions, which arise mainly fromdeamination, oxidation, etc. BER is initiated by excision of dam-aged bases, e.g. by DNA glycosylases. Human endonucleaseVIII-like protein 2 (hNEIL2) is a DNA glycosylase which prefersdamage in bubbles and other non-canonical DNA structures.Understanding the mechanisms of damage recognition andremoval, it is necessary to know the structural features of theenzyme. Among the attempts to study the structure of NEIL2proteins, only the opossum enzyme was crystallized and resolved.Using hydrogen deuterium exchange followed by mass spectro-metric analysis, we shown that in the free state the hNEIL2 pro-tein is in an open conformation. We also demonstrated that itcontains an extended disordered linker in the N-terminal domain(which makes crystallization nearly unfeasible). However, thestructure of the hNEIL2-DNA complex remains unclear. Weapplied a combination of HDX-MS and computer simulationmethods to study the structural and dynamic features of the for-mation of the human NEIL2 protein-substrate complex. Theexchange reaction was performed at 21°C and pH 7.6. The label-ing was terminated at time points between 10 s and 120 min byadding an aliquot of the reaction mixture to a quench buffer(0°C, pH 2.5) and immediately freezing the sample in liquidnitrogen. Immediately before analysis, the protein was thawedand subjected to cleavage on a column with immobilized pepsin.The peptides were retained on a trap column, concentrated anddesalted at 0°C, and then analyzed by MS. After processing thedata, we interpreted them by comparing them with the results ofcomputer simulations. This work was supported by grants fromthe RSF (20-14-00214).

UR - https://febs.onlinelibrary.wiley.com/doi/epdf/10.1002/2211-5463.13646

M3 - Conference article

VL - 13

SP - 172

JO - FEBS Open Bio

JF - FEBS Open Bio

SN - 2211-5463

IS - S2

M1 - P-06.1-08

ER -