{'fraction_non_alphanumeric': 0.024339720352149145, 'fraction_numerical': 0.02278612118073537, 'mean_word_length': 5.483221476510067, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Insights into the DNA cleavage mechanism of human LINE‐1 retrotransposon endonuclease

The human LINE‐1 endonuclease (L1‐EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1‐EN could help engineering them as genetic tools. We tested the in vitro activity of point mutants in three L1‐EN residues—Asp145, Arg155, Ile204—that are key for DNA cleavage, and determined their crystal structures. The L1‐EN structure remains overall unaffected by the mutations, which change the enzyme activity but leave DNA cleavage sequence specificity mostly unaffected. To better understand the mechanism of L1‐EN, we performed molecular dynamics simulations using as model the structures of wild type EN‐L1, of two βB6‐βB5 loop exchange mutants we have described previously to be important for DNA recognition, of the R155A mutant from this study, and of the homologous TRAS1 endonuclease: all confirm a rigid scaffold. The simulations crucially indicate that the βB6‐βB5 loop shows an anticorrelated motion with the surface loops βA6‐βA5 and βB3‐αB1. The latter loop harbors N118, a residue that alters DNA cleavage specificity in homologous endonucleases, and implies that the plasticity and correlated motion of these loops has a functional importance in DNA recognition and binding. To further explore how these loops are possibly involved in DNA binding, we docked computationally two DNA substrates to our structure, one involving a flipped‐out nucleotide downstream the scissile phosphodiester; and one not. The models for both scenarios are feasible and agree with the hypotheses derived from the dynamic simulations. The reduced cleavage activity we have observed for the I204Y mutant above however, favors the flipped out nucleotide model. Proteins 2009. © 2008 Wiley‐Liss, Inc.