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GC-Content Dependence of Elastic and Overstretching Properties of DNA:RNA Hybrid Duplexes.

ABSTRACT: DNA:RNA hybrid duplex plays important roles in various biological processes. Both its structural stability and interactions with proteins are highly sequence dependent. In this study, we utilize homebuilt optical tweezers to investigate how GC contents in the sequence influence the structural and mechanical properties of DNA:RNA hybrid by measuring its contour length, elasticities, and overstretching dynamics. Our results support that the DNA:RNA hybrid adopts a conformation between the A- and B-form helix, and the GC content does not affect its structural and elastic parameters obviously when varying from 40 to 60% before the overstretching transition of DNA:RNA hybrid occurs. In the overstretching transition, however, our study unravels significant heterogeneity and strong sequence dependence, suggesting the presence of a highly dynamic competition between the two processes, namely the S-form duplex formation (nonhysteretic) and the unpeeling (hysteretic). Analyzing the components left in DNA:RNA hybrid after the overstretching transition suggests that the RNA strand is more easily unpeeled than the DNA strand, whereas an increase in the GC content from 40 to 60% can significantly reduce unpeeling. Large hysteresis is observed between the stretching and relaxation processes, which is also quantitatively correlated with the percentage of unpeeling in the DNA:RNA duplex. Increasing in both the salt concentration and GC content can effectively reduce the hysteresis with the latter being more significant. Together, our study reveals that the mechanical properties of DNA:RNA hybrid duplexes are significantly different from double-stranded DNA and double-stranded RNA, and its overstretching behavior is highly sequence dependent. These results should be taken into account in the future studies on DNA:RNA-hybrid-related functional structures and motor proteins.

SUBMITTER: Yang D

PROVIDER: S-EPMC7451903 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.

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GC-Content Dependence of Elastic and Overstretching Properties of DNA:RNA Hybrid Duplexes.

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