Divalent metal ions, especially Mg2+, play pivotal roles in an enzyme’s ability to manipulate the highly stable structure of DNA. DNA and RNA polymerases, as well as numerous nucleases are clear examples of such enzymes, and are integral to critical cellular functions. Thus, these proteins represent important drug targets for various diseases and biotechnological tools for genome editing. Understanding the molecular mechanism of Mg2+-promoted DNA synthesis and cleavage is crucial for engineering and efficiently targeting of these enzymes. Time-resolved X-ray crystallography enables the visualization of catalytic processes and aids in dissecting catalytic molecular mechanisms. This technique tracks active conformations and intermediate states during catalysis by initiating chemical reactions in protein crystals synchronously via light activation or substrate diffusion. In my thesis work, I applied diffusion-based time-resolved crystallography to investigate two representative enzymes: DNA polymerase η, which is a Y-family DNA polymerase that participates during DNA replication to bypass bulky DNA lesions; and I-PpoI endonuclease, which is one-metal dependent His-Me nuclease that has an active site homologous to the HNH active site of the Cas9 nuclease. In elucidating the catalytic mechanism of DNA polymerases, wild-type and mutant variants of DNA polymerase η were generated and analyzed using kinetic assays. Over 100 crystal structures of DNA polymerase η complexed with a wide variety of deoxyribonucleotides, ribonucleotides, and nucleoside analogue drugs were determined with resolutions ranging from 1.4 to 2.8 Å, providing high-resolution visualization of canonical DNA synthesis and polymerase targeting. The structural alignments revealed the essential role of the third divalent metal ion and the dynamics of the primer end, including the sugar ring, correlated to substrate discrimination and efficient chemistry. Similarly, I tracked the reaction process of I-PpoI with kinetic assays and time-resolved crystallography. More than 40 crystal structures of I-PpoI at various pH and metal ion concentrations were determined. The intermediate structures revealed the involvement of one and only one divalent metal ion in DNA hydrolysis. DNA cleavage assays unveiled several possible deprotonation pathways for the nucleophilic water molecule. Importantly, metal ion binding and water deprotonation were found to be highly correlated during catalysis. These results offer mechanistic insights that can be instrumental in enhancing the bioengineering and targeting of polymerases and nucleases.