Giovanni Maga

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols—belonging to the B, Y and X classes—during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA polsu2009λ and η. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol λ, and 68-fold by DNA pol η, respectively. Experiments with DNA-pol-λ-null cell extracts suggested an important role for DNA polu2009λ. On the other hand, DNA polu2009ι, together with DNA polsu2009α, δ and β, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

Human DNA Polymerase λ Functionally and Physically Interacts with Proliferating Cell Nuclear Antigen in Normal and Translesion DNA Synthesis

Proliferating cell nuclear antigen (PCNA) has been shown to interact with a variety of DNA polymerases (pol) such as pol δ, pol ε, pol ι, pol κ, pol η, and pol β. Here we show that PCNA directly interacts with the newly discovered pol λ cloned from human cells. This interaction stabilizes the binding of pol λ to the primer template, thus increasing its affinity for the hydroxyl primer and its processivity in DNA synthesis. However, no effect of PCNA was detected on the rate of nucleotide incorporation or discrimination efficiency by pol λ. PCNA was found to stimulate efficient synthesis by pol λ across an abasic (AP) site. When compared with pol δ, human pol λ showed the ability to incorporate a nucleotide in front of the lesion. Addition of PCNA led to efficient elongation past the AP site by pol λ but not by pol δ. However, when tested on a template containing a bulky DNA lesion, such as the major cisplatin Pt-d(GpG) adduct, PCNA could not allow translesion synthesis by pol λ. Our results suggest that the complex between PCNA and pol λ may play an important role in the bypass of abasic sites in human cells.

DNA Polymerases: Discovery, Characterization and Functions in Cellular DNA Transactions

Maintenance of the information embedded in the genomic DNA sequence is essential for life. DNA polymerases play pivotal roles in the complex physiological processes of DNA replication and repair. Besides the tasks in vivo, DNA polymerases are the workhorses in numerous biotechniques such as polymerase chain reaction (PCR), cDNA cloning, genome sequencing, nucleic acids-based diagnostics, as well as techniques to analyze ancient and otherwise damaged DNA. The authors have recently witnessed the discovery of a plethora of novel DNA polymerases with specialized properties whose physiological functions are only just beginning to be understood. This book summarizes the current knowledge of these fascinating enzymes in viruses, bacteria, archaea and eukaryotes. Moreover, some diseases are related to DNA polymerase defects, and chemotherapy through inhibition of DNA polymerases is used to fight HIV, Herpes, as well as Hepatitis B and C infections. This book will appeal to a broad audience including basic scientists, diagnostic laboratories, and clinicians who will gain an invaluable understanding of these fascinating enzymes.

DNA Polymerases and Diseases

In this chapter, we would like to highlight the possible connections between DNA polymerases, the main enzymes in DNA metabolism, and human diseases, also critically evaluating those cases where the experimental data are not fully convincing. To this aim, we will first give a short overview of the three main DNA metabolic events, namely replication, repair and recombination, as well as of the checkpoint pathways acting in response to DNA damage. Besides a role in replication of the genome, DNA polymerases also have fundamental functions in other aspects of DNA metabolism, such as DNA repair, DNA recombination, translesion DNA synthesis and cell cycle checkpoint. In the last 10 years, numerous novel DNA polymerases have been revealed, but their exact cellular functions still await clarification. This review summarizes the known eukaryotic DNA polymerases and their relationships with human diseases.

Targeting Cellular Cofactors in HIV Therapy

Besides viral proteins cellular factors play a key role in the replication of the human immunodeficiency virus HIV-1. The outcome of virus replication is determined by the balance between the activity of a number of cellular dependency factors and restriction factors. Whereas the first are essential cofactors for diverse steps in the viral replication cycle, the latter counteract virus replication by sensing particular viral components as non-self, often as mediators of the innate immune system. Cellular cofactors include receptors for HIV-1 entry, LEDGF as cofactor for the viral integrase, the RNA helicase DDX3 involved in the nuclear export of unspliced viral RNAs, and diverse cellular kinases that promote viral replication. Cellular restriction factors are often antagonized by HIV-1 accessory proteins in order to counteract their restrictive function on viral replication. Although cellular cofactors in the HIV field are understood as factors promoting viral replication, we add a subchapter on the most important restriction factors (Trim5α, APOBEC3G, SAMHD1, and tetherin/BST-2). Today highly active antiretroviral therapy (HAART) mostly targets HIV proteins like reverse transcriptase, protease, or integrase to specifically interfere with virus replication. However, the identification of cellular cofactors and the increasing knowledge on their mode of action at defined steps in the HIV-1 replication cycle have opened new avenues towards the development of HIV-1 inhibitors. Here we summarize the most important cellular factors involved in HIV-1 replication along with therapeutic approaches developed to target them, preferentially without harming their normal cellular function.

Two Birds with a Stone: Molecular Cancer Therapy Targeting Signal Transduction and DNA Repair Pathways

The hallmarks of cancer cells are a higher proliferative activity and an aberrant genotype with respect to normal cells. These features can be exploited for the development of selective chemotherapeutic treatments against cancer. In particular, the connections among signal transduction pathways, cell cycle checkpoints and DNA replication and repair have the potential to provide new venues for the treatment of cancer. Here, we will review how the differences existing between normal and tumour cells, with respect to control of cell proliferation and maintenance of the genetic stability, can be exploited in cancer chemotherapy.