The Role of Base Excision Repair in Regulating DNA-Mediated Inflammatory Signaling Pathways

PROJECT SUMMARY Defects in DNA repair underlie a number of human genetic diseases that affect a wide variety of physiological systems and cause adverse phenotypes such as accelerated aging and predisposition to cancer. Faithful DNA repair is necessary to maintain genomic integrity and prevent cancer.

The base excision repair (BER) pathway is responsible for repairing at least 20,000 lesions per cell per day. If left unrepaired, the lesions can give rise to genomic instability and tumorigenesis.

BER is also the major repair pathway for nonbulky damaged bases, abasic sites, and DNA single-strand breaks after treatment with different DNA- damaging agents.

Several genes are involved in BER pathways, including DNA glycosylase, XRCC1, DNA polymerase beta (Pol?), DNA ligase III, flap endonuclease 1 (FEN1), and DNA ligase I.

BER deficiency can lead to accumulated unrepaired DNA damage, generating cytosolic DNA that likely activates DNA-dependent innate immune pathways.

Cyclic GMP-AMP synthase (cGAS) is a key cytosolic DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which triggers the activation of stimulator of interferon genes (STING), leading to type I Interferon production. However, how deficiency in BER promotes cGAS/STING-mediated inflammation is not yet fully understood.

The goal of this exploratory R21 proposal is to uncover how spontaneous DNA damage and failed BER stimulate host inflammatory response.

We recently developed a novel mouse model using a human genetic variant, which we will use to elucidate the molecular mechanism of inflammation.

Using a Cre-flox targeting system, we constructed an L22P conditional knock-in mouse model that lacks dRP lyase function expressed at Rosa26a locus and cannot support BER.

Our preliminary data revealed that BER deficiency in our mouse model markedly induces genomic instability and chronic inflammation.

Thus, we hypothesize that BER deficiency accumulates cytosolic DNA that derives from spontaneous DNA damage, thereby triggering the innate immune response.

In this study, we propose two Specific Aims: (1) Determine the molecular mechanisms through which aberrant BER induces inflammation.

Using BER-deficient cells ( Pol? -/-; XRCC1; PARP1-/-; L22P (dRP lyase-deficient Pol?), we will study how BER deficiency triggers innate immune response-mediated inflammation. (2) Determine whether failed BER stimulates cGAS/STING-mediated inflammation in mice.

Using in vitro and in vivo models (L22P mouse models), we will examine how cytosolic DNA-sensing pathways trigger inflammatory immune responses.

The outcomes of this study will define a novel paradigm for how aberrant BER induces the innate immune system, and will have broad implications for the molecular mechanisms behind cGAS/STING function, as well as for future immune-directed cancer therapies.