Question

1. Explain the mechanism of Mechanism of action of DNA polymerases.

2. Name any three replication accessory proteins. Explain how each function in DNA replication.

3. Describe Prokaryotic Transcription.

4. Describe Eukaryotic Transcription.

5. Briefly describe RNAi pathway for gene silencing.

6. Briefly describe RNA alternative splicing and RNA editing.

Answer 1

1. Mechanism of action of DNA polymerases

DNA polymerases are molecular motors directing the synthesis of DNA from nucleotides. All polymerases have a common architectural framework consisting of three canonical subdomains termed the fingers, palm, and thumb subdomains. Kinetically, they cycle through various states corresponding to conformational transitions, which may or may not generate force. In this review, we present and discuss the kinetic, structural, and single‐molecule works that have contributed to our understanding of DNA polymerase function.

The synthesis of DNA is catalyzed by an enzyme called DNA Polymerase. Unlike most enzymes, which have an active site dedicated to a single reaction, DNA polymerase uses a single active site  to catalyze addition of any of the four deoxynucleotide triphosphates. Polymerase shows kinetic selectivity, in which an enzyme favors catalysis using one of several possible substrates by dramatically increasing the rate of bond formation only when the correct substrate is present. Only when the correct pair is formed the catalysis occurs. Incorrect base pairing leads to dramatically lower rate of nucleotide addition due to the catalytically unfavorable alignment of these substrates. DNA polymerase show an impressive ability to discriminate between ribo- and deoxyribonucleoside phosphates. This ability is mediated by steric exclusion of rNTPs from the DNA polymerase active site, because in DNA polymerase the nucleotide binding pocket is too small to allow the presence of a 2’OH on the incoming nucleotide.

2. DNA replication accessory proteins provide particular functions that are mandatory for replicative pols. Such functions include the recruitment of particular pols when needed, the facilitation of pol binding to the primer terminus, the increase in pol processivity, the prevention of nonproductive binding of the pol to ssDNA, the release of the pol after DNA synthesis, and the bridging of pol interactions to other replication proteins. Thus, it is not surprising that these proteins are universally found in nature.

REPLICATION ACCESSORY PROTEINS

PCNA

Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by post-translational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities.

Replication factor C (RF-C)

Replication factor C (RF-C) is a five subunit DNA polymerase (Pol) δ/ε accessory factor required at the replication fork for loading the essential processivity factor PCNA onto the 3′-ends of nascent DNA strands. Here we describe the genetic analysis of the rfc2+ gene of the fission yeast Schizosaccharomyces pombe encoding a structural homologue of the budding yeast Rfc2p and human hRFC37 proteins. Deletion of the rfc2+ gene from the chromosome is lethal but does not result in the checkpoint-dependent cell cycle arrest seen in cells deleted for the gene encoding PCNA or for those genes encoding subunits of either Pol δ or Pol ε. Instead, rfc2Δ cells proceed into mitosis with incompletely replicated DNA, indicating that the DNA replication checkpoint is inactive under these conditions. Taken together with recent results, these observations suggest a simple model in which assembly of the RF-C complex onto the 3′-end of the nascent RNA-DNA primer is the last step required for the establishment of a checkpoint-competent state.

Replication protein A (RPA)

Replication protein A (RPA) is a highly conserved, eukaryotic ssDNA-binding protein essential for genome stability. RPA interacts with ssDNA and with protein partners to coordinate DNA replication, repair, and recombination. Single-molecule analysis of RPA–DNA interactions is leading to a better understanding of the molecular interactions and dynamics responsible for RPA function in cells. Here, we first describe how to express, purify, and label RPA. We then describe how to prepare materials and carry out single-molecule experiments examining RPA–DNA interactions using total internal reflection fluorescence microscopy (TIRFM). Finally, the last section describes how to analyze TIRFM data. This chapter will focus on human RPA. However, these methods can be applied to RPA homologs from other species.

3. Prokaryotic Transcription

Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring concurrently on the same DNA template. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein. Describing this process in Escherichia coli, a well-studied bacterial species. Although some differences exist between transcription in E. coli and transcription in archaea, an understanding of E. coli transcription can be applied to virtually all bacterial species.

4. Eukaryotic Transcription

Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus and organelles. With the genes enclosed in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes.

5. RNAi pathway for gene silencing

RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention, agriculture and other areas.

A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step, the trigger RNA (either dsRNA or miRNA primary transcript) is processed into an short, interfering RNA (siRNA) by the RNase II enzymes Dicer and Drosha. In the second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly and the single-stranded RNA hybridizes with mRNA target. Gene silencing is a result of nucleolytic degradation of the targeted mRNA by the RNase H enzyme Argonaute (Slicer). If the siRNA/mRNA duplex contains mismatches the mRNA is not cleaved. Rather, gene silencing is a result of translational inhibition.

6. RNA alternative splicing and RNA editing

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3' acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.