Figure 2: The DNA duplex AAA. The slashes indicate narrowing of the minor groove. Throughout the letters A, G, C and T are used to represent DNA nucleotide.

Sarma Laboratory

Professor of Chemistry, Ph.D., 1967, Brown University, Postdoctoral fellow, 1967-1970 Brandeis University, and University of California San Diego.
ph: 518-456-9362; fx: 518-452-3462; email: rhs07@albany.edu

Project 1: DNA Bending, Sugar Switch and Protein Binding
The research described below is being conducted in our laboratory in collaboration with Dr. Mukti H. Sarma at the University at Albany, Dr. Chris Turner at MIT, Dr. Victor Zhurkin at NIH, and Dr. G. Gupta at Los Alamos National Laboratory.

We aim to investigate our thesis that the free DNA that binds to the proteins in several systems such as TBP, HMG box domain, IHF, p53, CAP, etc come already locally deformed and bent. These local structural deformations are recognized by the DNA binding proteins, and used as a focal point to severely deform the DNA. Our thesis is based on own our demonstration that certain dinucleotde steps and DNA tracts (Figure 1) in free DNA display clearly distinct noncanonical B-form structures, and the sequences which the above proteins bind also contain many of the same or similar steps and tracts.We have detected and quantitated the significant presence of non-B-form motifs in dynamic equilibrium with the dominant B-form for the free duplexes.

Our conclusions come from an exhaustive study of the DNA duplex, code named AAA with the sequence shown in Figure 2. Our study consisted of determining reliably intimate details of the dynamic geometry of each of the 22 individual nucleotidyl units in AAA such as conformational population of the sugar ring, and that around the exocyclic linkages C3’-O3’, C4’-C5’ and C5’-O5’ from vicinal three bond and four bond 1H-1H, 1H-31P and 13C-31P coupling constants.We employed an array of antiphase 1H-1H and 1H-31P, and 1H-13C HSQC 2D NMR methods along with the computer simulation of such spectra to extract the coupling constants.

The data demonstrated the following:

At the 3’-end of the A-tract at the A-C•G-T step, the sugar switches from the south (C2’-endo type, B-form) to the north (C3’-endo type, A-form, Figure 3), leading to the local compression of the interphosphate distance, and consequent bending. In fact what we found was that the cytidine nucleotide in the duplex was C3’-endo, and its Watson-Crick pair guanosine, C2’-endo, creating a heteronomous pair at the 3’-end of the A-tract.



At the 5’-end of the A-tract, at the C-A•T-G step, the C4’-C5’ exocyclic linkage goes into trans conformation; in fact we reported that here that the T is trans whereas its Watson-Crick partner is gauche+ (Figure 4). This will lead to a local expansion of the interphosphate distance, allowing the rolling of the base pairs into the major groove leading to bending. A trans conformation for C4’-C5’ is not new. It was proposed by Watson and Crick back in 1953

The A-tract itself is polymorphic because as one travels towards the 5’-end of the duplex in the T-strand, the C4’-C5’ becomes increasingly trans, and the sugar pucker move toward C3’-endo.

We depict in Figure 5 qualitatively some of these features to realize how this creates distortion and bending in the DNA double helix.

Figure 5: A qualitative model of our sequence AAA showing the generation of local distortion and bending at the CA•TG step at the 5’-end of the A-tract, and at the AC•GT and CT•AG steps at the 3’-end of the A-tract due to change in C4’-C5’ torsion or switch in sugar pucker. The gray bars represent nucleotides in canonical B-form and thick broken yellow line is the B-DNA helix axis. The color bars represent nucleotides which can display local conformational variation, and the thin red line is the helix axis of segments at the end of A-tract. The helix axis bending angles at the 5' and 3' ends of the A-tract are provided by q1 and q2. At the 3' end of A-tract, the sugar of C8 assumes the A-form, thereby, shortening the local interphosphate distance. This shortening of the P-P distance will cause the edges of the adjacent bases to be closer to each other at the glycosidic linkage than at the Watson-Crick paired edges. This is the flaring or "rhombic" motif a la Zhurkin. At the 5' end of the A-tract, the effect is reverse, again limited to one strand. The trans conformation for C4'-C5' for T18 increases the P-P distance. This will cause the edges of the adjacent bases to be more distant from each other at the glycosidic linkage than at the Watson-Crick paired edges. This can be considered to be a "half butterfly" motif.

A note worthy feature of the diagram is that the upper part of the double helix (bases in gray) is in the canonical B-form, and the lower part (bases in color) contain all the deformations. This is because the deformations are limited to the T-strand at the 5' end and to the A-strand at the 3' end. This distribution of deformations in the same side of the helix axis (same face of DNA) may enable the transcription factor proteins to initially recognize them in an array.

The Sugar Switch.
The Importance of the Formation of a Patch of A-form within the matrix of B-DNA. Its Effect on DNA Bending and Protein Binding.
Prima facie one may think that a switch in one sugar out of 22 in our ideal 11-mer DNA duplex “AAA” from the south to the north conformation may have little or no influence on the overall DNA conformation and DNA bending. In Figure 6 we illustrate how a switch in C8 sugar from the south to the north affects the bending parameters as well as the overall conformation. The local roll increases by 5 to 13 degrees, tilt by 2 to 7 degrees with small changes in propeller twist and buckle. Particularly note worthy is the bend in the helix axis, and the significant displacement in the backbone.

Considerably more significant is the effect of the presence of a patch of A-form within the matrix of B-form DNA. Using the multiple copy refinement procedure which uses floating weights, recently published from James’ Lab at UCSF we have determined for our 11-mer DNA duplex (“AAA”) the populations and global structures of multiple conformers in an ensemble from NMR data of coupling constants and NOEs. The analysis clearly revealed that three global structures code named AAA_B-form, AAA_A-form_Patch I and AAA_A-form_Patch II had non-zero probabilities of 0.450, 0.393 and 0.157. To make it simple now, we show in Figure 7, these three forms schematically.

Figure 6a: A schematic representation of the structure and populations of three global structures of our 11-mer AAA duplex which contribute to the time average structure. The filled rectangles stand for B-form nucleotides, and the open rectangles for A-form nucleotides. Note that in the A-form_Patch I, with a population of 39%, the pair C8 and T9 at the 3’-end of the A-tract, and the pair T17 and T18 toward the 5’-end of the A-tract provide patches of A-forms to the DNA duplex. Note that the patches are heteronomous, that is, in the base pairs one nucleotide is C3’-endo, the other C2’-endo. In the A-form_Patch II, with a population of 15%, such heteronomous patches are extensive. The heteronomy results from the switch of pyrimidine nucleotides into the A-form. To our knowledge, this is the first time, the population distribution of DNA global structures has been experimentally determined by NMR spectroscopy. from J coupling and NOE data. (unpublished results).

What follows are graphical illustrations of the models of AAA_B-form, AAA_A-form_Patch I form (Figure 8), drawn from the coordinates generated by the multiple copy refinement program from the NMR data.They show with unmistakable clarity that these A-form patches contribute very significantly to DNA bending, and that they can provide a focal point for the binding of DNA binding proteins.

Figure 8: Color scheme: gray = AAA_B-form; cyan = A-form_Patch I; AAA tract = magenta; C3’-endo sugars = yellow. Left: In this stereo representation, the B-form and the A-form_Patch I structures are aligned along the AAA:TTT tract. Note that in the A-form_Patch I structure, the blue axis is bent into the major groove, at C4-A5 step. Right: Another stereo projection of the B-form and the A-form_Patch I structures aligned along the AAA:TTT tract. Note that here in the A-form_Patch I structure, the blue axis is bent into the minor groove, at A7-C8 step (unpublished data).

We project that such A-form patches will be present in the unbound DNA sequences where the architectural proteins bind into the minor groove. These patches which precipitate bending provide the initial recognition site, we postulate.

Why Should the Proteins Recognize Such A-form Patches within the Matrix of an otherwise B-Form ?
We hypothesize that the contacts between the protein side chains and the sugars are mostly hydrophobic. Apparently, these hydrophobic contacts in the minor groove stabilize the A-form of DNA in the complex, in addition to the hydrogen bonds between the DNA bases and the protein side chains in the major groove.

For this kind of interaction, it is important that the sugars are in C3'-endo conformation. Indeed, as we show (Figures 9) in our surface accessibility studies, the C2'-to-C3'-endo transition changes the orientation of the sugar rings, thereby exposing their CH and CH2 groups into the minor group.

Thus, even partial B-to-A transition in DNA [i.e., local switch in one or two sugars] increases the hydrophobicity of the DNA surface in the minor groove. We hypothesize that this effect is utilized by proteins interacting with the DNA sugars similar to the I-PpopI homing endonuclease or the human SRY protein.

Figure 9: Left Pair. Minor groove view of the NMR structure of d(AACTC):d(GAGTT). This is a pentamer duplex fragment from our 11-mer AAA duplex and from SRY human binding site. In panel 1, all DNA sugars are in 2'-endo conformation. In panel 2, conformation of the sugar of the central Cyt is switched to 3'-endo. This results in opening of the minor groove;the whole C3'-endo sugar (shown in green), and and O4' of Cyt (shown in yellow) become more accessible than in the case of C2'-endo (unpublished data).
Right Pair: Atom radii are extended by 2.5 Å to represent accessibility of the DNA surface to a probe of such a radius (e.g., end groups of Arg, Leu, Lys, etc.). Here, the difference between C2'- and C3'-endo sugars is even more clear. For example, in C2'-endo sugar the O4' atom is hidden by the opposite chain, whereas in the case of C3'-endo this atom is accessible for a large probe. Furthermore, the overall exposed surface of the sugar ring is larger for the C3'-endo puckering (unpublished data).

Significance
During the initiation of transcription, various transcription factors while in contact with the DNA filament, associate with each other over long distances of DNA via bending, deformation and looping of the intervening duplex). Several NMR and crystal structure studies of DNA-transcription factor complexes have revealed that in many of these instances, the proteins severely bend and deform the DNA.

But to truly understand this problem one must know both the starting and ending conformations of DNA. Despite the fact that, to date, structures of over 220 protein-DNA complexes have been reported, there are few cases in which high-resolution structures are available for DNA both free and in complex with the protein it binds.The reported NMR structures of free DNA sequences resembling transcription factor binding sites, such as A-tract sequences, are underdetermined time average structures which are always reported as B-form. Crystal structures of such sequences report them to be either bent due to artifacts of crystal packing or as straight. depending on various external factors which drive the crystal structures of DNA duplexes at the oligomer levels.

Professor Sarma’s Laboratory aims to provide the missing high resolution structures of several very important DNA sequences in the unbound state so that one could undertake a critical direct comparison of DNA structures before and after protein binding. Accurate determination of their structures in dynamic equilibrium (as opposed to time averaged), we believe, will show that they are already structurally “preformed” (preengineered) to recognize the protein, and bind to it. The local specific structural features engineered within the matrix of the DNA drives recognition and binding; it is not a direct readout of the base sequence. Sequences such the one involved in the binding of the master bender IHF will provide a wealth of information about local structural dynamics of several di- and trinucleotide steps, and what additional changes they undergo upon binding to a protein. One could use this new knowledge to predict anticipated structural changes in DNA sequences which bind to protein, but the structure of the complex itself is unknown. This predictive power is important because it is not possible to determine the structures of all DNA-protein complexes. Equally important is that in the long run, the information could be potentially valuable in the development of designer drugs to control expression of undesirable genes. For example, if this expression involves sequences which switch into A-form patches, a therapeutic agent which hydrates the DNA in the presence of the protein could be developed to suppress the expression. The therapeutic agent will be an antagonist to the protein which removes the water molecules from the minor groove, and thus propagates the A-form.

Project 2: Structure and Dynamics of a Peptide which inhibits breast cancer
In collaboration with Professors Anderson, Bennett and Jacobsen of Albany Medical College, Professor Sarma's laboratory is now investigating, using by High END NMR the structure and dynamics of a small peptide which inhibits growth of breast cancer.

Professor Sarma has some 150 research publications; in addition, he has edited over books and monographs in the discipline of biological structure, dynamics, interactions and expression. Below a few selected publications are provided.

Selected Publications

Sarma, M.H., Gupta, G., and Sarma, R.H., Structure of a Bent DNA: Two-Dimensional NMR Studies on d-GAAAATTTTC, Biochemistry, 27, 3423-3432 (l988).

Gupta, G., Sarma, M.H., and Sarma, R.H., On the Question of DNA Bending: Two-Dimensional NMR Studies on d-GTTTTAAAAC in Solution, Biochemistry 27, 7909-79l9, (l988).

Umemoto, K., Sarma, M.H., Gupta, G., and Sarma, R.H., Effect of Methyl Group on DNA Bending and Curvature: Structure of d(GA4U4C)2 in Solution, Biochemistry 29, 4714-4722 (1990).

Umemoto, K., Sarma, M.H., Gupta, G., Luo, J., and Sarma, R.H., Structure and Stability of a DNA Triple-Helix in Solution: NMR Studies on d(T)6.d(A)6.d(T)6 and its Complex with a Minor-Groove Binding Drug, Journal of the American Chemical Society 112, 4539- 4545 (1990).

Sarma, M. H., Umemoto, K., Gupta, G., Luo, J., and Sarma, R. H., In Search of a Hoogsteen Base Paired DNA in Aqueous Solution, J.Biomolecular Structure and Dynamics, 8, 461-482 (1991).

Ulyanov, N. B., Gorin, A. A., Zhurkin, V. B., Chen, B-C., Sarma, M. H., and Sarma, R. H., Systematic Study of Nuclear Overhauser Effects vis-a-vis Local Helical Parameters, Sugar Puckers, and Glycosidic Torsions in B DNA: Insensitivity of NOE to Local Transitions in B DNA Oligonucleotides Due to Internal Structural Compensations, Biochemistry 31, 3918-3930 (1992).

N. Ulyanov, M. H. Sarma, V. Zhurkin and R. H. Sarma, A Decreased interstrand H2’H1’ distance in the GC rich part of the duplex d(CTTCAAACTCC):d(GGAGTTTGAGG) in solution at low temperature: A proton nuclear magnetic resonance investigation. Biochemistry 32, 6875-6883 (1993).

Rajendra P. Ojha, Madan Mohan Dhingra, Mukti H. Sarma, Yash P. Myer, Robert F. Setlik, Massayuki Shibata, A. Latiff Kazim, Rick L. Orenstein, Robert Rein, Christopher J. Turner and Ramaswamy H. Sarma, Structure of an anti-HIV-1 Hammerhead Ribozyme Complex with a 17-mer DNA substrate analogue of HIV-1 gag RNA and a Mechanism for thr Cleavage Reaction: 750 Mhz NMR andComputer Experiments, J.Biomolecular Structure and Dynamics 15, 185-215 (1997).

Ramaswamy H. Sarma, Mukti H. Sarma, Linsen Dai, Kimiko Umemoto, GC -rich DNA Oligonucleotides with Narrow Minor Groove Width, FEBS Letters 418, 76-82 (1997)

Ojha, R. P., Dhingra, M. M., Sarma, M. H., Shibata, M., Farrar, M., Turner, C. J., and Sarma. R. H. DNA Bending and Sequence-Dependent Backbone Conformation: NMR and Computer Experiments, European Journal Of Biochemistry 265, 35-53 (1999)

Kamath, S., Sarma, M. H., Zhurkin, V. B., Turner, C. J., and Sarma, R. H., DNA Bending and Sugar Switching, J. Biomole.cular Structure and Dynamics, Conversation 11, 317-226 (2000).