       Document 0782
 DOCN  M94B0782
 TI    Analysis of strand transfer synthesis by reverse transcriptases.
 DT    9412
 AU    Buiser RG; Univ. of Rochester
 SO    Diss Abstr Int [B]; 54(7):3597 1994. Unique Identifier : AIDSLINE
       ICDB/94605826
 AB    Models of retrovirus replication require the viral reverse transcriptase
       (RT) to catalyze two distinct strand transfer reactions. In this
       process, an RNA primer is elongated on one template and then transferred
       to another template for additional elongation. It is thought that the RT
       ribonuclease H (RNase H) activity is involved in both transfer
       reactions. The properties of reverse transcriptases required for strand
       transfer synthesis have been examined using poly(rA) as a model
       substrate. In this system, strand transfer is observed by the appearance
       of products much longer than the templates used to generate them. RTs
       from human immunodeficiency (HIV), avian myeloblastosis (AMV), and
       murine leukemia (MuLV) viruses differ in molecular mass and subunit
       composition. However, they all catalyze strand transfer synthesis on
       poly(rA)300, generating characteristically long products. An RNase H
       deficient enzyme, HIV-RT(RD,) catalyzed strand transfer synthesis to the
       same degree as native HIV-RT, indicating that a functional RNase H
       activity is not required for this process. Additionally,
       N-ethylmaleimide (NEM), which inhibits RNase H but not polymerase
       activity of HIV-RT, did not diminish strand transfer synthesis. Highly
       processive DNA synthesis by each RT was found to be a requirement for
       the strand transfer reaction. Catalysis of strand transfer synthesis is
       not a property of all DNA polymerases, since the Klenow fragment of
       Escherichia coli DNA polymerase I is unable to catalyze this reaction.
       These results suggest that strand transfer synthesis relies on an
       unidentified functional activity present in RTs. The ability of RTs to
       catalyze strand transfer, or template switching, from internal regions
       of RNA templates has also been examined. To study this phenomenon, a
       system of donor and acceptor templates was employed in which homologous
       strand transfer can occur from a homopolymeric sequence positioned
       internally on the donor template. Our results indicate that HIV-, AMV-,
       and MuLV-RTs are all able to catalyze strand transfer from this internal
       homopolymeric sequence. Catalysis of this reaction is not dependent upon
       RNase H activity, since HIV-RT(RD) is able to catalyze the reaction
       efficiently. Additionally, NEM did not inhibit strand transfer by either
       the native or RNase H deficient forms of HIV-RT. Our data further
       indicate that template switching may be promoted by RT pausing at a
       specific site on the donor template. Conditions that increase RT pausing
       at this site also increase template switching. These results suggest
       that transient RT pausing at specific sites on the viral genome during
       reverse transcription may promote strand transfers that in turn lead to
       recombination. (Full text available from University Microfilms
       International, Ann Arbor, MI, as Order No. AAD93-34491)
 DE    DNA Polymerase I/GENETICS  Ethylmaleimide/PHARMACOLOGY  *Reverse
       Transcriptase/*GENETICS  Ribonucleases/*GENETICS  THESIS

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

