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Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus.

Montaño SP, Coté ML, Roth MJ, Georgiadis MM - Nucleic Acids Res. (2006)

Bottom Line: In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3' to the CA dinucleotide step, resulting in a reactive 3' OH on one strand and a 5' two base overhang on the complementary strand.The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice.This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3' and 5' flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202, USA.

ABSTRACT
In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3' to the CA dinucleotide step, resulting in a reactive 3' OH on one strand and a 5' two base overhang on the complementary strand. In order to investigate the structural properties of the 3' end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3' and 5' flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

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(A) Crystal structure of a 16mer DNA duplex in complex with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. The ribbon-rendering was done with MOLSCRIPT (41). The DNA structure shown here is that of the LTR-A. The asymmetric unit in this lattice contains one protein molecule and one-half the DNA molecule. The dashed line marks the crystallographic 2-fold axis. (B) The LTR-A, -B, -C and -D sequences for which we have determined crystal structures are shown. These sequences are derived from the 3′-processing site of the actual LTR. The bases underlined are those in common with the actual LTR sequence. In LTR-B, the eight base-pairs that are boxed in is the LTR-LTR junction and outlined with a darker box is the CA cut site. The numbering scheme for the bases is shown in the LTR-A sequence. (C) The structures of LTR-A (orange), -B (blue and red), -C (black) and -D (green) are shown as stick renderings. Arrows indicate the site of cleavage immediately 3′ of the CA dinucleotide step.
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fig1: (A) Crystal structure of a 16mer DNA duplex in complex with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. The ribbon-rendering was done with MOLSCRIPT (41). The DNA structure shown here is that of the LTR-A. The asymmetric unit in this lattice contains one protein molecule and one-half the DNA molecule. The dashed line marks the crystallographic 2-fold axis. (B) The LTR-A, -B, -C and -D sequences for which we have determined crystal structures are shown. These sequences are derived from the 3′-processing site of the actual LTR. The bases underlined are those in common with the actual LTR sequence. In LTR-B, the eight base-pairs that are boxed in is the LTR-LTR junction and outlined with a darker box is the CA cut site. The numbering scheme for the bases is shown in the LTR-A sequence. (C) The structures of LTR-A (orange), -B (blue and red), -C (black) and -D (green) are shown as stick renderings. Arrows indicate the site of cleavage immediately 3′ of the CA dinucleotide step.

Mentions: The host-guest crystallographic method takes advantage of minor groove binding interactions of the N-terminal fragment of the MMLV RT to the ends of duplex DNA. This method facilitates crystallization and analysis of DNA sequences of interest as previously described (19–22,28,29). Briefly, the interactions of the duplex DNA are limited to the minor groove and backbone of the terminal 3 bp of a 16 bp duplex, which is bound on either end by an N-terminal fragment molecule (see Supplementary Data and Table S1 for a full description of protein–DNA interactions). Thus, the middle 10 bp of the 16 bp duplex are free of interactions with the protein. The asymmetric unit, the unique repeating unit of the crystal, includes one protein molecule and one half of the 16 bp duplex. The 16 bp duplex is bisected by a crystallographic 2-fold rotation axis, as shown in Figure 1A. As a consequence of this symmetry, the electron density observed is an average of the two halves of the DNA molecule. Although we have successfully used this method to analyze asymmetric DNA sequences (21), the method is better suited to the analysis of symmetric sequences.


Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus.

Montaño SP, Coté ML, Roth MJ, Georgiadis MM - Nucleic Acids Res. (2006)

(A) Crystal structure of a 16mer DNA duplex in complex with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. The ribbon-rendering was done with MOLSCRIPT (41). The DNA structure shown here is that of the LTR-A. The asymmetric unit in this lattice contains one protein molecule and one-half the DNA molecule. The dashed line marks the crystallographic 2-fold axis. (B) The LTR-A, -B, -C and -D sequences for which we have determined crystal structures are shown. These sequences are derived from the 3′-processing site of the actual LTR. The bases underlined are those in common with the actual LTR sequence. In LTR-B, the eight base-pairs that are boxed in is the LTR-LTR junction and outlined with a darker box is the CA cut site. The numbering scheme for the bases is shown in the LTR-A sequence. (C) The structures of LTR-A (orange), -B (blue and red), -C (black) and -D (green) are shown as stick renderings. Arrows indicate the site of cleavage immediately 3′ of the CA dinucleotide step.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636480&req=5

fig1: (A) Crystal structure of a 16mer DNA duplex in complex with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. The ribbon-rendering was done with MOLSCRIPT (41). The DNA structure shown here is that of the LTR-A. The asymmetric unit in this lattice contains one protein molecule and one-half the DNA molecule. The dashed line marks the crystallographic 2-fold axis. (B) The LTR-A, -B, -C and -D sequences for which we have determined crystal structures are shown. These sequences are derived from the 3′-processing site of the actual LTR. The bases underlined are those in common with the actual LTR sequence. In LTR-B, the eight base-pairs that are boxed in is the LTR-LTR junction and outlined with a darker box is the CA cut site. The numbering scheme for the bases is shown in the LTR-A sequence. (C) The structures of LTR-A (orange), -B (blue and red), -C (black) and -D (green) are shown as stick renderings. Arrows indicate the site of cleavage immediately 3′ of the CA dinucleotide step.
Mentions: The host-guest crystallographic method takes advantage of minor groove binding interactions of the N-terminal fragment of the MMLV RT to the ends of duplex DNA. This method facilitates crystallization and analysis of DNA sequences of interest as previously described (19–22,28,29). Briefly, the interactions of the duplex DNA are limited to the minor groove and backbone of the terminal 3 bp of a 16 bp duplex, which is bound on either end by an N-terminal fragment molecule (see Supplementary Data and Table S1 for a full description of protein–DNA interactions). Thus, the middle 10 bp of the 16 bp duplex are free of interactions with the protein. The asymmetric unit, the unique repeating unit of the crystal, includes one protein molecule and one half of the 16 bp duplex. The 16 bp duplex is bisected by a crystallographic 2-fold rotation axis, as shown in Figure 1A. As a consequence of this symmetry, the electron density observed is an average of the two halves of the DNA molecule. Although we have successfully used this method to analyze asymmetric DNA sequences (21), the method is better suited to the analysis of symmetric sequences.

Bottom Line: In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3' to the CA dinucleotide step, resulting in a reactive 3' OH on one strand and a 5' two base overhang on the complementary strand.The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice.This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3' and 5' flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202, USA.

ABSTRACT
In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3' to the CA dinucleotide step, resulting in a reactive 3' OH on one strand and a 5' two base overhang on the complementary strand. In order to investigate the structural properties of the 3' end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3' and 5' flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

Show MeSH
Related in: MedlinePlus