Enzymes of Nucleic Acid Synthesis and Modification

Volume 1: DNA Enzymes

Samson T. Jacob

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351088756
• Edition: 1
• Topic: Medizin
• Subtopic: Biochemie in der Medizin

The First volume gives an overview of the enzymes involved in DNA synthesis and modification; the second volume deals with the RNA-enzymes. Although the major emphasis of the book is on eukaryotic enzymes, a separate chapter dealing with prokaryotic DNA repair enzymes has been included to discuss the major advances in this field in recent years. There are two separate chapters on RNA polymerases to provide a comprehensive coverage of the enzymes from lower eukaryotes, plants and higher eukaryotes.

Chapter 1

REVERSE TRANSCRIPTASE

Gary F. Gerard

TABLE OF CONTENTS

• I. Introduction
  • A. Scope
  • B. Background
    • 1. Life Cycle of Retroviruses
    • 2. The RNA of Retroviruses
    • 3. Structural Proteins of Retroviruses
• II. Biosynthesis of Pol Gene Products
  • A. Synthesis
  • B. Posttranslational Processing
    • 1. Proteolytic Cleavage Patterns and Nature of Mature Products
    • 2. Phosphorylation
    • 3. Involvement of Viral-Coded Protease
    • 4. Site of Processing
• III. Enzymatic and Nucleic Acid Binding Activities of Pol Gene Products
  • A. DNA Polymerase
    • 1. Comparison with Other DNA Polymerases
    • 2. Mode of Action
  • B. RNase H
    • 1. Discovery and General Properties
    • 2. Mode of Action
    • 3. Relationship to DNA Polymerase
  • C. DNA Endonuclease
    • 1. Reverse Transcriptase-Associated DNA Endonuclease
    • 2. Other Pol Gene-Coded DNA Endonuclease Activities
  • D. Nucleic Acid Binding Activities
    • 1. Binding to tRNA
    • 2. Unwinding-Like Activity
• IV. Genetic Evidence for the Role of Pol Gene Products in the Life Cycle of Retroviruses
• V. Function of Pol Gene Products in Retroviral DNA Synthesis and Integration
  • A. DNA Synthesis
    • 1. A Perspective
    • 2. Final Products of DNA Synthesis
    • 3. Synthesis of (−) Strand DNA
    • 4. Synthesis of (+) Strand DNA
  • B. Integration
• Acknowledgments
• References

I. Introduction

A. Scope

Retrovirus reverse transcriptase has been the subject of intense investigation since its discovery in 1970.^1,2 Over the years, a large number of reviews have appeared treating the expanse of data published concerning the enzyme (Table 1). Recent discoveries indicate that in addition to the RNase H and DNA polymerase activities of reverse transcriptase, the polymerase gene of retroviruses codes for a DNA endonuclease activity which may or may not be found associated with reverse transcriptase. This chapter will focus on the enzymatic and nucleic acid binding properties of these proteins and the role they play in the replication of retroviruses, and will attempt to place this analysis in perspective by making comparisons, where possible, with other better understood replicative systems.

Retroviruses and their resident reverse transcriptases have been characterized from many different vertebrates. Most of our knowledge concerning reverse transcriptase and the replication of retroviruses is derived from studies with avian leukosis-sarcoma viruses (ALSV) and the enzyme from the avian myeloblastosis virus (AMV) complex of viruses, and to a lesser extent with murine leukemia virus (MuLV) and Moloney MuLV reverse transcriptase. This discussion will be confined to these two virus systems, which should portray a general picture applicable to most retroviruses and their enzymes.

B. Background

In order to understand the role of reverse transcriptase in the retrovirus life cycle a certain amount of background is essential.

1. Life Cycle of Retroviruses

The first event in the retrovirus life cycle is adsorption of the virion to the cell surface, followed by penetration of the virion into the cell cytoplasm. Uncoating of the virus particle to expose the internal core^20-23 probably takes place within the cytoplasm the first 1 to 2 hr after infection.²⁴ Viral RNA in the core is transcribed within the cytoplasm into linear duplex DNA^16,25-27 which is transported to the nucleus where it can be converted to covalently closed circular DNA.²⁸ Mature duplex DNA is integrated into the host genome.^29,30 The mechanism of integration and the structure of the precursor to integration are not yet established. Subsequently, the integrated viral DNA is transcribed by cellular RNA polymerase³¹ and transcripts are processed and transported to the cytoplasm. Viral mRNA is translated into a series of precursor polyproteins which encapsidate 35S viral RNA³² and a specific subset population of cell tRNAs³³ and the maturing particles move to the cell surface, from which they bud and acquire an outer envelope.³² Protein products of the retrovirus polymerase gene are specifically involved in RNA to DNA synthesis, encapsidation of specific cell tRNAs, and perhaps in viral DNA integration.

2. The RNA of Retroviruses

The 50 to 70S genomic RNA of replication competent retroviruses contains two identical subunits of about 35S (molecular weight of 2.7 to 3.0 × 10⁶) and smaller (4 to 5S) RNA species.¹⁴ The RNA in the 50 to 70S complex as isolated in a protein-free state from virus has extensive secondary and tertiary structure involving hydrogen bonding between complementary bases within individual 35S subunits³⁴ as well as between subunits.³⁵ Based upon electron microscopic analysis under partially denaturing conditions,³⁶ the most stable secondary structural features of the genome RNA complex include (1) a dimer linkage between 35S subunits involving fewer than 50 nucleotides approximately 450 nucleotides from their 5′ ends,^36,37 (2) a large loop in each subunit positioned 2 to 4 kilobases (Kb) from the 5′ end,^36,37 and (3) a large hairpin involving some 300 nucleotides located 70 nucleotides from the 5′ end of each subunit.³⁷ It appears that formation of these relatively stable structures requires a special environment present only during virion budding and maturation,^38,39 since the structures do not reform in vitro after denaturation.^36,37 That reverse transcriptase can translocate these regions and other potential regions^34,35 of RNA secondary structure during DNA synthesis is probably the result of its ability to catalyze strand displacement synthesis (Section III.A.1.) and perhaps because of the involvement of nucleic acid binding proteins which reduce the secondary structure of RNA (Section I.B.3). In addition, the presence of two identical subunits in a dimer structure provides the opportunity for reverse transcriptase to use either one or both subunits in generating a final DNA product, particularly since the final double-stranded (ds) DNA product contains repeated sequences not repeated in subunit RNA. Whether one or both subunits are involved has not been established.

Table 1
REVIEWS DEALING WITH REVERSE TRANSCRIPTASE

Emphasis	Ref.
Properties, functions, and structures of reverse transcriptase in viruses and cells	3—9
Reverse transcriptase in cells and properties distinguishing it from other DNA polymera...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Contributors
• Chapter 1 Reverse Transcriptase
• Chapter 2 Eukaryotic DNA Polymerases
• Chapter 3 Terminal Deoxynucleotidyl Transferase
• Chapter 4 DNA Methylases
• Chapter 5 Prokaryotic DNA Repair Enzymes
• Chapter 6 Mammalian DNA Repair Enzymes
• Index