RecQ helicases are ubiquitous DNA unwinding enzymes, essential in the maintenance of genome stability by acting at the interface between DNA replication, recombination and repair. Humans have five different paralogues of RecQ helicases namely RecQ1, BLM, WRN, RecQ4 and RecQ5. Germ-line mutations in the recq4 gene give rise to three distinct human genetic disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency and predisposition to cancer. Moreover, RecQ4 sporadic mutations are implicated in many types of cancer, including osteosarcoma and lymphoma. At the same time, overexpression of RecQ4 in tumour development and progression has been observed in breast, cervical and prostate cancer. In addition to the central helicase core, RecQ4 has a unique N-terminal domain, which is essential for viability and has homology to the yeast Sld2 replication initiation factor, followed by a Zn knuckle, and an uncharacterized C-terminal domain. The role of these accessory regions in the function of the RecQ4 helicase was assessed by a combination of biochemical and biophysical experiments. Many RecQ helicases have a C-terminal domain which folds into a helical bundle known as the Helicase and RnaseD-like C-terminal domain (HRDC). Despite the lack of sequence homology, a detailed bioinformatics analysis led us to predict that the RecQ4 C-terminal domain may fold as a HRDC domain. We expressed and characterized the C-terminal domain of human RecQ4. CD spectra analysis of the recombinant fragment does suggest it folds into a helical bundle, consistent with our prediction. We show that the domain does not bind nucleic acid substrates. To further investigate its possible role in assisting the unwinding, we tested the ability of the helicase domain, alone and in trans with the C-terminal domain, to unwind a variety of DNA and RNA structures and found that the presence of the C-terminal domain enhances in vitro unwinding for a variety of substrates, especially for R-loops, the substrate most efficiently unwound by RecQ4, as our colleagues have previously shown (unpublished experiments). We have also produced a number of C-terminal site-directed mutants, either based on sequence conservation or present in Rothmund-Thomson patients, and tested the effect of the mutations on the biochemical activity. Our experiments indicate that the mutation of all selected residues (except Arg1162) seem to affect the R-loops resolving activity of the helicase core of RecQ4, and especially the RTS patient mutations, which cause a dramatic reduction in the activity of the protein. We also investigated the effect of an N-terminal region encompassing the end of the Sld2 homologous region and the Zn knuckle. This region does bind all the substrates with a preference for R-loops, Holliday Junctions, D-loops and hybrid fork-RNA/DNA and its presence, in trans with the helicase core domain of the protein, strongly enhances RecQ4 binding to nucleic acids. Moreover, our experiments show that its presence significantly increases the unwinding activity of the helicase core towards all the substrates, especially for R-loops. To enhance our understanding of the activity of the N-terminal domain towards R-loops unwinding and resolution, a number of mutants within the zinc knuckle, and predicted and/or shown to be functionally important in nucleic acid interaction, were produced and used in R-loops unwinding assays. Two mutants (Asn406Cys and in particular Phe404Ala/Trp412Ala) showed a significant decrease in R-loops resolving activity of the protein, suggesting a role for these residues in substrate interaction. This complex scenario would suggest a role of N-terminal domain as a major nucleic acid interacting region of the protein, increasing the unwinding affinity towards all the substrates, while the C-terminal domain is an essential player in imparting substrate specificity to the protein, promoting R-loops resolution. These results enrich our knowledge about RecQ4 structure, function and suggests novel roles of RecQ4 in DNA replication and genome stability. Moreover, since misregulation of R-loops might cause neurological diseases and cancer, a detailed biochemical and structural analysis of human RecQ4 and its role in R-loops metabolism may also shed light on these devastating diseases.

The role of the accessory N- and C-terminal domains in modulating the helicase activity of human RecQ4 / Bagnano, Ilenia. - (2019 Oct 29).

The role of the accessory N- and C-terminal domains in modulating the helicase activity of human RecQ4

Bagnano, Ilenia
2019-10-29

Abstract

RecQ helicases are ubiquitous DNA unwinding enzymes, essential in the maintenance of genome stability by acting at the interface between DNA replication, recombination and repair. Humans have five different paralogues of RecQ helicases namely RecQ1, BLM, WRN, RecQ4 and RecQ5. Germ-line mutations in the recq4 gene give rise to three distinct human genetic disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency and predisposition to cancer. Moreover, RecQ4 sporadic mutations are implicated in many types of cancer, including osteosarcoma and lymphoma. At the same time, overexpression of RecQ4 in tumour development and progression has been observed in breast, cervical and prostate cancer. In addition to the central helicase core, RecQ4 has a unique N-terminal domain, which is essential for viability and has homology to the yeast Sld2 replication initiation factor, followed by a Zn knuckle, and an uncharacterized C-terminal domain. The role of these accessory regions in the function of the RecQ4 helicase was assessed by a combination of biochemical and biophysical experiments. Many RecQ helicases have a C-terminal domain which folds into a helical bundle known as the Helicase and RnaseD-like C-terminal domain (HRDC). Despite the lack of sequence homology, a detailed bioinformatics analysis led us to predict that the RecQ4 C-terminal domain may fold as a HRDC domain. We expressed and characterized the C-terminal domain of human RecQ4. CD spectra analysis of the recombinant fragment does suggest it folds into a helical bundle, consistent with our prediction. We show that the domain does not bind nucleic acid substrates. To further investigate its possible role in assisting the unwinding, we tested the ability of the helicase domain, alone and in trans with the C-terminal domain, to unwind a variety of DNA and RNA structures and found that the presence of the C-terminal domain enhances in vitro unwinding for a variety of substrates, especially for R-loops, the substrate most efficiently unwound by RecQ4, as our colleagues have previously shown (unpublished experiments). We have also produced a number of C-terminal site-directed mutants, either based on sequence conservation or present in Rothmund-Thomson patients, and tested the effect of the mutations on the biochemical activity. Our experiments indicate that the mutation of all selected residues (except Arg1162) seem to affect the R-loops resolving activity of the helicase core of RecQ4, and especially the RTS patient mutations, which cause a dramatic reduction in the activity of the protein. We also investigated the effect of an N-terminal region encompassing the end of the Sld2 homologous region and the Zn knuckle. This region does bind all the substrates with a preference for R-loops, Holliday Junctions, D-loops and hybrid fork-RNA/DNA and its presence, in trans with the helicase core domain of the protein, strongly enhances RecQ4 binding to nucleic acids. Moreover, our experiments show that its presence significantly increases the unwinding activity of the helicase core towards all the substrates, especially for R-loops. To enhance our understanding of the activity of the N-terminal domain towards R-loops unwinding and resolution, a number of mutants within the zinc knuckle, and predicted and/or shown to be functionally important in nucleic acid interaction, were produced and used in R-loops unwinding assays. Two mutants (Asn406Cys and in particular Phe404Ala/Trp412Ala) showed a significant decrease in R-loops resolving activity of the protein, suggesting a role for these residues in substrate interaction. This complex scenario would suggest a role of N-terminal domain as a major nucleic acid interacting region of the protein, increasing the unwinding affinity towards all the substrates, while the C-terminal domain is an essential player in imparting substrate specificity to the protein, promoting R-loops resolution. These results enrich our knowledge about RecQ4 structure, function and suggests novel roles of RecQ4 in DNA replication and genome stability. Moreover, since misregulation of R-loops might cause neurological diseases and cancer, a detailed biochemical and structural analysis of human RecQ4 and its role in R-loops metabolism may also shed light on these devastating diseases.
ONESTI, Silvia Caterina Elvira
Bagnano, Ilenia
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11767/103933
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