The central dogma of molecular biology, which involves replication, transcription and translation, describes the flow of gene expression from DNA to RNA to protein. DNA converts genetic information by transcribing RNA through RNA polymerase (RNAP) and then translating protein by ribosomes. To maintain both efficiency and fidelity during the transmission of genetic information to proteins, many enzymes are involved. In this thesis, the working mechanism of two enzymes will be discussed: DNA glycosylase and RNAP. DNA glycosylase excises lesions from DNA to maintain the integrity of the genome and RNAP transcribes from DNA into RNA.

Alkylpurine glycosylase D (AlkD), a DNA glycosylase, is responsible for repairing DNA damage to maintain the integrity of the genome. However, how gly...[ Read more ]

The central dogma of molecular biology, which involves replication, transcription and translation, describes the flow of gene expression from DNA to RNA to protein. DNA converts genetic information by transcribing RNA through RNA polymerase (RNAP) and then translating protein by ribosomes. To maintain both efficiency and fidelity during the transmission of genetic information to proteins, many enzymes are involved. In this thesis, the working mechanism of two enzymes will be discussed: DNA glycosylase and RNAP. DNA glycosylase excises lesions from DNA to maintain the integrity of the genome and RNAP transcribes from DNA into RNA.

Alkylpurine glycosylase D (AlkD), a DNA glycosylase, is responsible for repairing DNA damage to maintain the integrity of the genome. However, how glycosylases efficiently and accurately recognize DNA lesions among numerous base pairs has not been elucidated. It has been hypothesized that glycosylase translocation occurs by alternating between a high speed-low accuracy diffusion mode and a low speed-high accuracy mode in searching for lesions. A slow mode exists when AlkD approaches lesions and is thus important for lesion searching. However, the molecular mechanism for the slow mode has not been determined. In this study, using the Markov state model built from extensive all-atom Molecular dynamics (MD) simulations, we elucidated the mechanism governing the slow mode. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. In addition, essential roles of Y27 and Q38 in diffusion were identified.

RNAP facilitates DNA transcription of RNA. Transcription includes initiation, elongation and termination. Previous studies have provided crystal structures for intermediate states in the initiation process. However, due to the large conformational changes exhibited by the RNAP-DNA complex, the kinetic mechanism of this complex during initiation has not been elucidated. In this study, coarse-grained MD was applied to study the initiation of the RNAP-DNA complex. Our results suggest that the flexibility of critical regions, including fork loop 2 and sigma fingers, gave space to the template DNA entering the active site.